A Colloidal Drug Delivery System for Antiallergic Drug

A Colloidal Drug Delivery System for Antiallergic Drug

A Thesis submitted to Gujarat Technological University

for the Award of

Doctor of Philosophy

in

Pharmacy

by

Ms. Jaswandi Udhav Girme

[Enrollment No. : 119997290027]

under supervision of

Dr. Naazneen Surti

GUJARAT TECHNOLOGICAL UNIVERSITY

AHMEDABAD

December - 2019

ii

© Jaswandi Girme

iii

DECLARATION

I declare that the thesis entitled ―A Colloidal Drug Delivery System for Antiallergic Drug”

submitted by me for the degree of Doctor of Philosophy is the record of research work carried

out by me during the period from July 2011 to Dec 2019 under the supervision of Dr. Naazneen

Surti and this has not formed the basis for the award of any degree, diploma, associateship,

fellowship, titles in this or any other University or other institution of higher learning.

I further declare that the material obtained from other sources has been duly acknowledged in

the thesis. I shall be solely responsible for any plagiarism or other irregularities, if noticed in

the thesis.

Signature of the Research Scholar: Date: 21/7/2020

Name of Research Scholar: Ms. Jaswandi U. Girme

Place: Vadodara, Gujarat, India.

iv

CERTIFICATE

I certify that the work incorporated in the thesis ―A Colloidal Drug Delivery System for

Antiallergic Drug” submitted by Ms. Jaswandi U. Girme was carried out by the candidate

under my supervision/guidance. To the best of my knowledge: (i) the candidate has not

submitted the same research work to any other institution for any degree/diploma,

Associateship, Fellowship or other similar titles (ii) the thesis submitted is a record of original

research work done by the Research Scholar during the period of study under my supervision,

and (iii) the thesis represents independent research work on the part of the Research Scholar.

Signature of Supervisor: Date: 21/7/2020

Name of Supervisor: Dr. Naazneen Surti


v

Originality Report Certificate

It is certified that PhD Thesis titled ―A Colloidal Drug Delivery System for Antiallergic

Drug” by Ms. Jaswandi U. Girme has been examined by us. We undertake the following:

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under consideration to be published elsewhere. No sentence, equation, diagram, table,

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vii

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viii

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Place: Vadodara, Gujarat, India

Seal

ix

(briefly specify the modifications suggested by the panel)

(The panel must give justifications for rejecting the research work)

Thesis Approval Form

The viva-voce of the PhD Thesis submitted by Ms. Jaswandi U. Girme (Enrollment No.

119997290027) entitled ―A Colloidal Drug Delivery System for Antiallergic Drug” was

conducted on Tuesday, 21/7/2020 (day and date) at Gujarat Technological University.

(Please tick any one of the following option)

The performance of the candidate was satisfactory. We recommend that he/she be awarded

the PhD degree.

Any further modifications in research work recommended by the panel after 3 months

from the date of first viva-voce upon request of the Supervisor or request of Independent

Research Scholar after which viva-voce can be re-conducted by the same panel again.

The performance of the candidate was unsatisfactory. We recommend that he/she should not be awarded the PhD degree.

Dr. Naazneen Surti Dr. Brahmeshwar Mishra

Name and Signature of Supervisor with Seal 1) (External Examiner 1) Name and Signature

Dr. Chandrakant Kokare

2) (External Examiner 2) Name and Signature

X

X

√

x

ABSTRACT

Allergic conjunctivitis (AC), one of the most eyesight-threatening infection, defined by ocular

itching, hyperemia, lacrimation and edema, impairs the quality of life across the globe.

Ebastine is available as an oral antihistamine formula for allergic disorders, such as tablets and

syrup. Oral ebastine causes unfavorable effects on heart like QT prolongation, severe gastric

distress, decreased tear production, resulting in dryness of the ocular surface, which

exacerbates ocular discomfort and increases susceptibility of eye to irritation. Topical

antihistamines are preferred for treating ocular allergies over oral agents since their direct

application at the site of action results in rapid onset and superior efficacy with less systemic

side effects. Hence, topical formulation of ebastine was developed to achieve its onsite

exposure for ocular allergies. Moreover, conjunctiva is more accessible to hydrophilic

molecules than lipophilic molecules. This creates challenge for a lipophilic molecule such as

ebastine for topical ocular development. Successful dissolution of ebastine in o/w

microemulsion allows its use in more convenient soluble form. Initially, solubility of drug in

various oils, surfactant and cosurfactant was determined, followed by pseudo-ternary phase

diagram to find microemulsion area. The D-optimal mixture design was employed for

optimization of formulation. The optimized microemulsion formulation was characterized for

its transparency, pH, drug content, droplet size, zeta potential, viscosity, osmolarity, refractive

index and surface tension etc. The UV spectrophotometric analytical method was used for the

in vitro analysis of drug. Bioanalytical method was developed and validated for ebastine for

assessing pharmacokinetic parameters of the drug in ocular tissue matrix and plasma. The

Campul MCM EP was selected as oil phase and blend of Labrasol with Tween 80 and

Propylene glycol with glycerol were used as Smix. The resultant optimized microemulsion

formulation showed droplet size of 142 ± 0.16 nm, polydispersity index below 1, refractive

index 1.369 ± 0.04 and osmolarity 291 ± 0.301 mOsm/L. The pH value of the developed

formulation was 6.9 ± 0.12, which can be easily buffered by tear fluid (pH 7.2-7.4). The

surface tension of the developed formulation was found to be 34.75 ± 0.13 mN/m. Low

surface tension ensures good spreading effect on ocular surface and mixing with precorneal

film components, thereby improving contact with ocular surface. Zeta potential and viscosity

of developed formulations was found to be -22.6 ± 0.39 mV and 13.19 ± 0.121cps

xi

respectively. The optimum physicochemical properties were found to be eye-fitting. Carboxy

methyl cellulose and sodium hyaluronate were used as gelling agents at different

concentrations to prepare microemulsion based gel with a goal of increasing residential time at

the site of action. The ebastine microemulsion based gel was evaluated for various parameters

like pH, rheological assessment, drug content, mucoadhesive strength and spreadability. The

antiallergic potential of optimized formulation was assessed by performing in vitro study like

hen's egg chorioallantoic membrane test (HET-CAM) for tolerability and in vivo efficacy

study in ovalbumin (OA)-induced allergic conjunctivitis (AC) with acute ocular irritation

study and blinking index. Eye scratching behavior and edema were evaluated after topical

antigen challenge followed by histopathology. The results showed that developed formulation

was effective in inhibiting symptoms of eye inflammation induced by ovalbumin. Acute

ocular irritation test was performed using rabbits and results showed that developed

formulation was non-irritant to the eye. The in-vivo pharmacokinetic studies revealed

increased concentration of the drug in ocular tissue matrix with negligible systemic

absorption. The study revealed that the developed formulation of ebastine was retained at

ocular site up to 8 hr., showing ocular tissue concentration in multiples of IC 50 (oral) reported

for antihistaminic and rest of late phase allergy mediator‘s antagonistic action. Hence,

prepared microemulsion based gel of ebastine has great potential for treating early as well as

late allergy symptoms and can be considered as an great alternative to customary oral

formulations of poorly soluble antiallergic drug, exhibiting site specific delivery.

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ACKNOWLEDGEMENT

The satisfaction that accompanies the successful completion of any task would be incomplete without

mentioning the peoples who made it possible and whose constant guidance and encouragement crown

all the efforts with success. It is not a fair task to acknowledge all the peoples who made this PhD

thesis possible with few words, However I will try to do my best to extend my great appreciation to

everyone who helped me scientifically and emotionally throughout this journey. Although, it is just my

name on the cover, many peoples have contributed to the research in their own particular way and for

that I want to give them special thanks.

I offer prayer of thanksgiving to the almighty God for being the source of strength throughout the years

of my research work to reach this day of success.

With a deep sense of respect and gratitude, I would like to express my sincere thanks to my esteemed

guide, Dr. Naazneen Surti, Professor & Vice- Principal, Babaria Institute of Pharmacy, Vadodara, for

giving me the opportunity to work in the field of colloidal drug delivery. Without her perpetual

encouragement, constructive, prompt, timely and helpful guidance throughout my journey of the

doctoral research, I would never have succeeded in accomplishing the work.

With great reverence, I take this opportunity to express my debt of gratitude to the DPC (Doctoral

Progress committee) members, Dr. M.C. Gohel, research director, Anand Pharmacy College, Anand,

Gujarat & consultant, Alembic Pharmaceuticals Ltd. and Dr. Tejal Mehta, Professor & Head, Institute of

Pharmacy, Nirma University, Ahmadabad for providing their valuable insights, their critical review,

guidance and support during the work.

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I wish to express my heartiest thanks Dr. Vimal Kumar, Dean, ITM School of Pharmacy, ITMBU

University, Dr. Abhay Dharamsi, Dean, Faculty of Pharmacy, Parul University, Dr. Umesh

Upadhay, Principal, Sigma Institute of Pharmacy, Dr. Falguni Tandel, Head of the Pharmaceutical

Analysis department, Parul University, Dr. Shweta Gupta, Associate Professor, Parul University, Dr.

Asha Patel, Associate Professor, Parul University, for providing me the infrastructural and other

direct or indirect support for the successful completion of research work.

I am immensely thankful to Mr. Sameer Mehetre, Senior manager, Drug discovery, SPARC, Mr.

Prashant Bahekar, Senior, manager, Drug discovery, SPARC, Dr. Gajanan Shinde, Senior manager,

F&D, Mil Laboratories Pvt. Ltd for their kind help in data analysis.

It would be remiss on my part if I don’t acknowledge the help and support of all my friends especially

Dr. Prachi Karia, research scholar, M.S. University, Ms. Meenakshi Sharma and other colleagues

for their wonderful company, unending inspirations, motivations, constant encouragement, technical

assistance throughout the research work.

I am also thankful to SICART (Sophisticated Instrumentation Centre for Applied Research and

Testing) for providing me the technical and infrastructural support for carrying out the DSC analysis,

TEM analysis.

I wish to express my sincere thanks to all editors, publishers and their honorable referees who have

published our research papers.

I am immensely thankful to M/s Bal Pharma Pvt. Ltd, Bommasandra, Bangalore, India, for providing

the gift sample of drug, M/s Gattefosse, Saint-Priest, France, M/s Abitec Corp, Ohio, USA for

providing the gift samples of excipients for the study, Government Poultry House, Vadodara for

providing fertilized eggs for CAM assay.

I would also like to put in record my thanks to Honorable Vice Chancellor Prof. (Dr.) Navin Sheth,

Ex-Vice-Chancellor Dr. C.N. Patel, BOG member, Dr. Rakesh Patel, BOG member Mr. J.C. Lilani,

Research Coordinator, Ms. Mona Chaurasiya, Mr. Dhaval Gohil and other Staff Members of Ph.D

section for their co-operative assistance and support.

At the last, I would like to thank my daughter for kind support and patience showed by her during the

years of PhD. I apologize to my daughter, Sanvi for not giving her time; I am amazed with the level of

understanding shown by her during my research work. And finally, I would like to thank my truly

amazing husband, Mr. Sameer Mehetre, whose love and encouragement allowed me to finish this

journey. He supported me every step of the way with patience, great insight, humor and knowledge.

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Gratitude is not just an action, not just saying thank you to animate object, few inanimate objects are

also paramount significant like my shelter, my untired laptop, my unwater logged plants n rest of all

to whom I should apologize……….

Thanks to one & all… Ms. Jaswandi u. Girme

Date: December, 2019

Place: Vadodara

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Dedicated to my Beloved

Family Members

& The Saraswati Mata…

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Table of Contents

S.No. Contents P. No.

i Title Page. i

ii Declaration iii

iii Certificate iv

iv Originality Report Certificate v

v Non-Exclusive License Certificate. vii

vi Thesis Approval Certificate ix

vii Abstract x

viii Acknowledgement xii

ix Dedication page xv

x Table of Contents xvi

xi List of Abbreviation xxiv

xii List of Symbols xxvii

xiii List of Figures xxix

xiv List of Tables xxxii

xv List of Appendices xxxv

Chapter 1 Introduction 1-13

1.1 Definition of Problem 3

1.2 Aim of the Research Work 4

1.3 Objective and Scope of Research Work 5

1.4 Scope of Research Work 5

1.5 Rationale of Research Work 5

1.5.1 Rational for Selection of Ocular Route of Drug Delivery 5

1.5.2 Rationale for Selection of Microemulsion Drug Delivery System 6

1.5.3 Rationale for Selection of Drug 7

1.6 Proposed Plan of Work 7

1.7 Original Contribution by the Thesis 8

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1.8 Outline of Thesis 8

1.9 References 9

Chapter 2 Literature Review 14-55

2.1 Overview 15

2.2 Allergic Conjunctivitis 17

2.2.1 Types of Allergic Conjunctivitis 17

2.2.2 Complications of Allergic Conjunctivitis 21

2.2.3 Treatment of Allergic Conjunctivitis 22

2.3 Microemulsion as Colloidal Drug Delivery for Ocular Therapy 25

2.3.1 Anatomy & Physiology of Eye as Barriers for Ocular Drug

Delivery

25

2.3.2 Conventional Vs. Colloidal Drug Delivery Platform 26

2.3.3 Microemulsion 27

2.3.4 Theories of Microemulsion Formation 28

2.3.5 Components of Microemulsion 29

2.4 Literature Survey 32

2.5 Drug and Excipients Profile 35

2.5.1 Ebastine 35

2.5.2 Campul MCM EP 36

2.5.3 Tween 80 37

2.5.4 Labrasol 38

2.5.5 Glycerol 39

2.5.6 Propylene glycol 41

2.5.7 Sodium Hyaluronate 42

2.5.8 Carboxy methyl cellulose 43

2.5.9 Sodium perborate 44

2.6 References 45

Chapter 3 Materials and Methods 56-93

3.1 Material and Equipment Used 57

3.2 Identification of Drug 59

xviii

3.2.1 Physical Appearance of Drug 60

3.2.2 Melting Point 60

3.2.3 Solubility 60

3.2.4 FT-IR Study 60

3.2.5 Differential Scanning Calorimetry 61

3.3 Analytical Method 61

3.3.1 Estimation of Ebastine using UV Spectrophotometry 61

3.3.1.1 Calibration Curve of Ebastine in Methanol as a Solvent 61

3.3.1.2 Calibration Curve of Ebastine in Methanolic Phosphate

Buffered Saline (PBS, pH 7.4, 30% v/v) 62

3.3.2 High Performance Liquid Chromatography (HPLC) Method Development 63 and Validation

3.3.2.1 Analytical Method Development 64

3.3.2.2 Analytical Method Validation 66

a. Linearity and Range 66

b. Precision 66

c. Accuracy 66

d. Sensitivity 67

e. Extraction Recovery 67

f. Robustness 67

g. Stability 68

h. System Suitability 68

i Statistical Analysis 68

3.4 Screening of Components of Microemulsion Formulation17

69

3.4.1 Selection of Oil 69

3.4.2 Selection of Surfactants 69

3.4.3 Selection of Co-surfactants 70

3.4.4 Drug Excipients Compatibility Study 70

3.5 Construction of Pseudo- Ternary Phase Diagrams 70

3.6 Preparation of Microemulsion 71

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3.7 Optimiza tion of Microemulsion by D-Optimal Mixture Design 72

3.7.1 Preparation of drug loaded microemulsion 73

3.8 Formulat ion and Optimization of Microemulsion Based Gel 73

3.9 Evaluatio n of Optimized Microemulsion Formulation 74

3.9.1 Measurement of pH 74

3.9.2 Droplet size, Zeta potential and Viscosity measurement 74

3.9.3 Measurement of Refractive Index 74

3.9.4 Measurement of Osmolarity 75

3.9.5 Measurement of Surface Tension 75

3.9.6 Determination of Drug Content 75

3.9.7 Transmission Electron Microscopy 75

3.9.8 Measurement of % Transmittance 76

3.10 Evaluati on of Microemulsion Based Gel 76

3.10.1 Measurement of pH 76

3.10.2 Rheology study 76

3.10.3 Mucoadhesive strength 76

3.10.4 Spreadability 77

3.10.5 Drug Content 77

3.10.6 In vitro Drug Release Study 77

3.10.7 Kinetics of Drug Release Study 78

3.10.7.1 Zero Order Release Equation 78

3.10.7.2 First Order Release Equation 78

3.10.7.3 Higuchi Square Root of Time Model 78

3.10.7.4 Korsmeyer-Peppas Model 79

3.11 Steriliza tion and Sterility Testing 80

3.11.1 Sterilization 80

3.11.2 Sterility Testing 80

3.12 In vitro / In vivo studies 80

3.12.1 Ocular Irritation Study by Hen‘s Egg Chorioallantoic Membrane

(HET-CAM) Test

81

3.12.2 Ocular Tolerability Study by Blinking index 81

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3.12.3 Acute Ocular Irritation Study 82

3.12.4 Efficacy Study by Ovalbumin Induced Allergic Conjunctivitis

Model

82

3.12.4.1 Edema Scoring 83

3.12.4.2 Scratching Behavior 83

3.12.5 Histopathological Study 83

3.13 Pharmacokinetic Study 84

3.14 Stability Studies 84

3.14.1 Accelerated Stability Study by Centrifugation Stress Test 84

3.14.2 Stability Study as per ICH guidelines 84

3.15 Data Analysis 85

3.16 References 85

Chapter 4 Results and Discussion 90-180

4.1 Identification of Drug 91

4.1.1 Identification of Drug by Physical Attributes 91

4.1.2 Identification of Drug by FTIR 91

4.1.3 Identification of Drug by DSC 94

4.2 Analytical Method 94

4.2.1 UV Spectrophotometric Estimation of Ebastine 95

4.2.1.1 Calibration Curve in Methanol as Solvent 95

4.2.1.2 Calibration Curve in Methanolic Phosphate Buffer (PBS, pH 97 7.4 , 30% v/v) as solvent

4.2.2 HPLC Method Development and Validation 98

4.2.2.1 RP- HPLC Method development for Pure Ebastine 98

a. System Suitability Test 99

b. Linearity and Range 100

c. Repeatability 101

d. Precision 102

e. Accuracy 103

f. Limit of Detection (LOD) and Limit of Quantification

(LOQ) 104

g. Robustness 104

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e

4 .2.2.2 Bioanalytical Method Development in Ocular Tissue 105

a. System Suitability Test for Drug with Internal Standard 107


c. Extraction Trials in Ocular Tissue 110

d. System Suitability Test for Drug with Internal Standard

in Ocular Tissue 111

e. Sensitivity 112

f. Linearity and Range 113

g Accuracy 115

h. Precision 115

i. Recovery 116

j. Stability Study 118

4 .2.2.3 Bioanalytical Study of Plasma Sample 121

a. System Suitability Test for the Drug with Internal

Standard in Plasma 122


4.3 Screening of Components of Microemulsion 125

4.3.1 Sel ection of Oil 125

4.3.2 Sel ection of Surfactant 127

4.3.3 Sel ection of Co-surfactant 128

4.3.4 Dr ug Excipient Compatibility Study 130

4.3.5 Dif ferential Scanning Calorimetry Study 131

4.4 Constructi on of Pseudo- ternary Phase Diagrams 132

4.5 Optimizati on of Microemulsion by D-Optimal Mixture Design 136

4.5.1 Ex perimental Validation of Design Space 145

4.6 Formulatio n and Optimization of Microemulsion Based Gel 146

4.7 Evaluation of Optimized Microemulsion Formulation 148

4.7.1 pH 148

4.7.2 Dr oplet size, Zeta Potential and Viscosity Measurement 148

4.7.3 M asurement of Refractive Index 150

4.7.4 Me asurement of Osmolarity 150

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4.7.5 Measurement of Surface Tension 151

4.7.6 Drug Content 151

4.7.7 Transmission Electron Microscopy 151

4.7.8 Measurement of % Transmittance 152

4.8 Evaluati on of Microemulsion Based Gel 152

4.8.1 pH 152

4.8.2 Rheology Study 153

4.8.3 Mucoadhesive Strength 156

4.8.4 Spreadability 156

4.8.5 Drug content determination 156

4.8.6 In vitro Drug Release Study 156

4.8.7 Kinetics of Drug Release Study 158

4.9 Steriliza tion and Sterility Testing 161

4.10 In vitro / In vivo studies 163

4.10.1 Ocular Irritation Study by Hen‘s Egg Chorioallantoic Membrane (HET-

CAM) Test 163

4.10.2 Ocular Tolerability Study by Blinking index 165

4.10.3 Acute Ocular Irritation Study 165

4.10.4 Efficacy Study by Ovalbumin Induced Allergic Conjunctivitis Model 166

4.10.4.1 Edema scoring 166

4.10.4.2 Scratching Behavior 169

4.11 Histopa thological Study 169

4.12 Pharma cokinetic Study 171

4.13 Stability Studies 173

4.13.1 Accelerated Stability Test by Centrifugation Stress Test 173

4.13.2 Stability study as per ICH guidelines 173

4.14 Referenc es 176

Chapter 5 Summa ry and Conclusion 181-189

5.1 Summar y of the Work 182

5.2 Achieve ment with Respect to the Objective 188

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5.3 Conclusion 188

Appendices

190-191

Appendix I: Approval Certificate from CPCSEA & IAEC Committee for

Animal Study

190

Appendix II: Dose Calculation Method 191

List of Publications 192

xxiv

List of Abbreviations

Abbreviations Full form

AC Allergic conjunctivitis

SAC Seasonal allergic conjunctivitis

PAC Perennial allergic conjunctivitis

VKC Vernal keratoconjunctivitis

AKC Atopic keratoconjunctivitis

GPC Giant papillary conjunctivitis

BCS Biopharmaceutical classification system

ICAM Intercellular adhesion molecule

VCAM Vascular cell adhesion molecule

MCP Monocyte chemo attractant protein

IL Interleukin

MIP Macrophage inflammatory protein

TAB Tablet

SOLN Solution

OD Once daily

BID Twice daily

BW Body weight

CMC Critical micelle concentration

HLB Hydrophilic lipophilic balance

IOP Intraocular pressure

ME Microemulsion

BRB Blood retinal barrier

BAB Blood aqueous humor Barrier

CFR Code of federal regulations

CPCSEA Committee for the Purpose of Control and Supervision of Experiments on Animals

Da Dalton

DLS Dynamic light scattering

DoE Design of experiment

EBA Ebastine

xxv


FDA Food and drug administration

PEG Polyethylene glycol

GIT Gastro- Intestinal Tract

GRAS Generally regarded as safe

HED Human equivalent dose

HPLC High performance liquid chromatography

RP-HPLC Reversed Phase - high Performance liquid chromatography

UV Ultra-violet

FT-IR Fourier transform infrared

IAEC Institutional animal ethics committee

ICH International council for harmonization

IUPAC International union of pure and applied chemistry

IV Intra-venous

LLOQ Lower Limit of quantification

LOD Limit of detection

LOQ Limit of quantification

QC Quality control

LQC Low quality control

MQC Medium quality control

HQC High quality control

% CV Percentage coefficient of variance

FT Freeze thaw

ISTD Internal standard

ANOVA Analysis of variance

RT Room temperature

%RSD Percentage relative standard deviation

SD Standard deviation

NMT Not more than

PBS Phosphate-buffered Saline

PDI Poly dispersity index

HET-CAM Hen‘s egg chorioallantoic membrane

B.I. Blinking index

xxvi


OA Ovalbumin

KCS Keratoconjunctivitis sicca

S.E.M. Standard error of the mean

DPX Mixture of distyrene,plasticizer,xylene

AUC Area under plasma concentration curve

AUMC Area Under the First moment of the concentration

MRT Mean residential time

Cmax Maximum plasma concentration

tmax Time for maximum plasma concentration

PK Pharmacokinetic

IVIVC In-vivo In-vitro correlation

ADME Adsorption, distribution, metabolism, elimination

K2EDTA Dipotassium ethylenediaminetetraacetic acid

EPP Eppendorf tube

FTGM Fluid thioglycolate medium

SCDM Soyabean casein digest medium

CR Controlled release

v/s Verses

DSC Differential scanning calorimetry

HCl Hydrochloric Acid

CMC Carboxy methyl cellulose

SH Sodium hyaluronate

RH Relative humidity

RSM Response surface methodology

TEM Transmission electron microscopy

USFDA United states food and drug administration

USP United states pharmacopoeia

DPX Mixture of distyrene,plasticizer,xylene

WHO World health organization

RPM Rotations per minute

SD Sprague dawley

xxvii

List of Symbols

Symbol Meaning

hr. Hour

s-1 Per second

min Minute

µg Microgram

gm Gram

mg Milligram

ng Nanogram

cm Centimeter

nm Nanometer

± Positive or Negative

λmax Maximum wavelength

°C Degree Celsius

°F Degree Fahrenheit

K Kelvin

mV Millivolts

R2 Linear correlation coefficient

T Time

% Percent

mM Milimole

Q Amount of drug released

K0 Zero order release constant

K1 First order release constant

Kh Higuchi square root of time release constant

Q∞ Total drug released after infinite time

n Diffusion exponents that characterizes the

mechanism of drug release

Qt Drug released at time t

kh Higuchi Square Root constant

kKP Korsmeyer-Peppas constant

KDa Kilo Dalton

v/w Volume/weight fraction

w/w Weight/weight fraction

gm/ml Gram /milliliter fraction

Pka Degree of ionization

Psi Pounds per square inch

xxviii

Symbol Meaning

Conc. Concentration

pH Potential hydrogen

pKa Acid dissociation constant

Smix Mixture/Blend of surfactant &co-surfactant

ppm Parts per million

γ Shear strain

𝜏 Shear stress

M Torque

Pa Pascal

cP Centipoise

xxix

List of Figures

Figure Caption P. No.

2.1 Schematic representation of allergic cascade in allergic conjunctivitis 16

2.2 Seasonal and perennial allergic conjunctivitis 18

2.3 Vernal keratoconjunctivitis 19

2.4 Atopic keratoconjunctivitis 20

2.5 Giant papillary conjunctivitis 20

2.6 Complications of allergic conjunctivitis 21

2.7 Technology platforms for ocular therapeutics 22

2.8 Treatment / Medication of allergic conjunctivitis 23

2.9 Market scenarios for treatment of allergic conjunctivitis 23

2.10 Schematic representation of visionary organ ―Eye‖ 26

4.1 (a) Observed IR spectra of ebastine 92

4.1 (b) Reference IR spectra of ebastine 92

4.2 Structure of ebastine 93

4.3 DSC Thermogram of ebastine 94

4.4 (a) Overlay spectra of ebastine at different concentrations 96

4.4 (b) Calibration curve for ebastine in methanol as solvent 96

4.5 (a) Overlay spectra of ebastine at different concentrations 97

4.5 (b) Calibration curve for ebastine in in methanolic PBS (pH 7.4, 30% v/v) as

solvent 98

4.6 Chromatogram of system suitability 100

4.7 (a) Chromatogram for linearity study 101

4.7 (b) Calibration plot for linearity study 101

4.8 (a) Chromatogram of pheniramine maleate (ISTD) 106

4.8 (b) Chromatogram of phenylephrine hydrochloride (ISTD) and ebastine 106

4.8 (c) Chromatogram of phenylephrine Hydrochloride (ISTD) and ebastine in

tissue matrix 107

4.9 Chromatogram of system suitability of ebastine and ISTD 107

xxx

4.10 (a) Chromatogram for linearity study of drug with ISTD 109

4.10 (b) Calibration plot for linearity study of drug with ISTD 110

4.11 Chromatogram of drug with ISTD in ocular tissue 111

4.12 Chromatogram of system suitability of drug with ISTD in ocular tissue 112

4.13 (a) Overlay chromatogram for linearity study of drug with ISTD in ocular

tissue 114

4.13 (b) Calibration plot for linearity study of drug with ISTD in ocular tissue 115

4.14 Chromatogram of drug with ISTD in plasma 122

4.15 Chromatogram of system suitability of drug with ISTD in plasma 122

4.16(a) Overlay chromatogram for linearity study of drug with ISTD in plasma 124

4.16(b) Calibration plot for linearity study of drug with ISTD in plasma 125

4.17 Solubility of ebastine in different oils 126

4.18 Solubility of ebastine in different surfactants 128

4.19 Solubility of ebastine in different co-surfactants 129

4.20 IR spectra for drug excipient compatibility 130

4.21 DSC Thermogram of physical mixture (drug + polymers) 131

4.22 (a) Pseudo Ternary Diagram of Oil: Smix individual system (1:1), (2:1), (3:1) 135

4.22 (b) Pseudo Ternary Diagram of Oil: Smix Blend system 2(1:1):1, 2(1:1):

1(1:1). 135

4.23 (a) Response variable globule size (Y1) 140

4.23 (b) Response variable viscosity (Y2) 142

4.23 (c) Response variable % transmittance (Y3) 144

4.24 Overlay plot 145

4.25 IR Spectra for drug excipient compatibility 148

4.26 Globule size measurement of optimized formulation 149

4.27 Zeta potential measurement of optimized formulation 150

4.28 Transmission Electron Microscopy (TEM) of optimized formulation 152

4.29 (a) Rheogram of microemulsion based gel 154

4.29 (b) Rheogram of microemulsion based gel diluted with tear fluid 155

4.30 In vitro release profiles of microemulsion and microemulsion based gel 157

xxxi

4.31 (a) Models for drug release kinetics, Zero order release kinetics (Microemulsion) 159

4.31 (b) Models for drug release kinetics, First order release kinetics

(Microemulsion) 159

4.31 (c) Models for drug release kinetics, Higuchi Model of release kinetics

(Microemulsion) 159

4.31 (d) Models for drug release kinetics, Korsemeyer - Pepppa‘s Model of release

kinetics (Microemulsion) 159

4.32 (a) Models for drug release kinetics, Zero order release kinetics

(Microemulsion based gel) 160

4.32 (b) Models for drug release kinetics, First order release kinetics

(Microemulsion based gel)x`x 160

4.32 (c) Models for drug release kinetics, Higuchi Model of release kinetics

(Microemulsion based gel) 160

4.32 (d) Models for drug release kinetics, Korsemeyer - Pepppa‘s Model of release

kinetics (Microemulsion based gel) 160

4.33 Sterility testing of optimized microemulsion formulation using soybean-

casein digest medium and fluid thioglycolate medium 162

4.34 Ocular irritation study by Chorioallantoic Membrane Test 164

4.35 Acute ocular irritation study 165

4.36(a) Effect of optimized formulation on ovalbumin-induced conjunctivitis in

guinea pigs, Time point: 0.5 hr. 167

4.36(b) Effect of optimized formulation on ovalbumin-induced conjunctivitis in

guinea pigs, Time point: 24 hr. 167

4.37 Edema response 168

4.38 Scratching response 169

4.39 Histopathological photomicrographs of the conjunctival tissues in

ovalbumin induced allergic conjunctivitis 170

4.40 Fitting the experimental data to Pharmacokinetic Model (PK Solver 2.0) 171

xxxii

List of Tables

Table Caption P. No.

2.1 List of antiallergic drugs used in the treatment of allergic conjunctivitis 24

2.2 List of components used in the ocular microemulsion system. 31

2.3 Recent studies on the microemulsion system for the treatment of

glaucoma 32

2.4 Recent studies on the microemulsion system for the ocular delivery of

bacterial keratitis 33

2.5 Recent studies on microemulsion system for the treatment of uveitis. 33

2.6 Recent studies on microemulsion for the ocular delivery of

immunosuppressant 34

3.1 List of materials utilized during research work 57

3.2 List of equipment utilized during research work 59

3.3 Values of diffusional exponent and corresponding release mechanism 79

4.1 Identification test for ebastine with the standard/ inference 91

4.2 Interpretation of FTIR spectra of ebastine 93

4.3 Calibration curve data for ebastine in methanol as solvent 95

4.4 Calibration curve data for ebastine in methanolic PBS (pH 7.4, 30% v/v)

as solvent

97

4.5 Trials for selection of mobile phase 99

4.6 Data of system suitability for ebastine (10 µg/ml) 100

4.7 Data of repeatability 102

4.8 Data of interday precision 102

4.9 Data of intraday precision 103

4.10 Data of accuracy 103

4.11 Data of robustness at different wavelength 104

4.12 Different experimental trials for RP-HPLC method development 106

4.13 System suitability parameters of chromatogram for ebastine and ISTD 108

4.14 (a) Sample preparation for linearity studies of drug with ISTD 108

xxxiii

4.14 (b) Concentration, area and area ratio for linearity study of drug with ISTD 109

4.15 Data obtained for extraction trials in ocular tissue 111

4.16 System suitability parameters of drug with ISTD in ocular tissue 112

4.17 Sensitivity data for LLOQ samples 113

4.18 Concentration, area and area ratio for linearity study of drug with ISTD

in ocular tissue

124

4.19 Data of accuracy for ebastine 115

4.20 Data of precision for ebastine 116

4.21 (a) Data of recovery at LQC 117

4.21 (b) Data of recovery at MQC 117

4.21 (c) Data of recovery at HQC 118

4.22 (a) Data of short term stability 119

4.22 (b) Data of freeze thaw stability 119

4.22 (c) Data of long term stability 120

4.22 (d) Data of stock solution stability at room temperature after 6 hours 120

4.22 (e) Data of stock solution stability at refrigerated condition (2-8˚C) for 7

days

121

4.23 Data of system suitability parameters of ebastine with ISTD in plasma 123

4.24 Concentration, area and area ratio for linearity study of drug with ISTD

in plasma

124

4.25 Selection of oil based on solubility of drug in oil 126

4.26 Selection of surfactant based on solubility of drug in surfactant 127

4.27 Selection of co-surfactant based on solubility of drug in co-surfactant 129

4.28 (a) Trial compositions of preliminary pseudo ternary batches of Oil: Smix

Individual system

132

4.28 (b) Trial compositions of preliminary pseudo ternary batches of Oil: Smix

Blend system

134

4.29 Compositions of design matrix batches with responses 136

4.30 Coefficients of Cubic equation for each independent variable 137

4.31 Summary of regression analysis for all responses 138

xxxiv

4.32(a) Data of ANOVA table for globule size (Y1) 139

4.32(b) Data of ANOVA table for viscosity (Y2) 141

4.32(c) Data of ANOVA table for % transmittance (Y3) 143

4.33 Checkpoint analysis of optimized formulation 145

4.34 Different trial batches for selection of gelling/ mucoadhesive polymers 147

4.35 Different trials batches for microemulsion based gel 147

4.36 Regression coefficients for release kinetics 160

4.37 (a) Results of sterility testing in FTGM (Fungi) 161

4.37 (b) Results of sterility testing in SCDM (Bacteria) 161

4.38 Blinking index and clinical symptoms 165

4.39 Edema scoring (At 0.5 h and 24 h after Topical Antigen Challenge) 166

4.40 Single dose pharmacokinetic study parameters 172

4.41 (a) Stability study data for microemulsion formulation 174

4.41 (b) Stability study data for microemulsion based gel formulation 175

xxxv

List of Appendices

Appendix I: Approval Certificate from CPCSEA & IAEC Committee for Animal Study

Appendix II: Dose Calculation Method

Chapter 1 Introduction

119997290027 / Gujarat Technological University Page 1

CHAPTER 1

Introduction



Allergic diseases like atopic dermatitis, contact dermatitis, conjunctivitis, allergic rhinitis and

rhinoconjuctivitis are a major public health burden worldwide. As the prevalence of these dis-

eases is increasing, novel and improved therapies become prime need. Conjunctivitis is a

global disease encountered in clinical practice all over the world especially higher rate of in-

fection was found in developing countries. Recent studies suggest that up to 40% of the popu-

lation in developed countries experience symptoms of allergic conjunctivitis (AC) and endure

vision threatening ailments that offer a lower quality of life. They can range from ocular dis-

comfort to vision loss. The ophthalmic medical product market continues to grow at a strong

pace, from approximately $30 billion in 2016 to an estimated $42 billion by 2023 registering

compound annual growth rate of 5.3 from 2017 to 2023.1

Allergic conjunctivitis (AC), defined by ocular itching, hyperemia, lacrimation and edema. It

is caused by inappropriate response of the ocular surface to various environmental allergens.

As most of the inflammation affects the conjunctiva, the word "allergic conjunctivitis" is in-

terchangeably used with ocular allergies. The different types of ocular allergies include pre-

dominantly ocular itch-inducing seasonal allergic conjunctivitis (SAC) and perennial allergic

conjunctivitis (PAC) to more severe sight-threatening vernal keratoconjunctivitis (VKC) and

atopic keratoconjunctivitis (AKC).2-4

The pathophysiology of allergic cascade include early

phase symptoms and late phase responses.5 Current therapy available in market becoming the

drug of choice for treating only immediate early phase symptomatic relief for patients with

allergic conjunctivitis. On this background, necessity emerges for selection of drug acting on

early as well as late phase of allergy cascade as well as designing formulation strategies with

desired ocular attributes.

Colloidal drug delivery systems have been widely studied and explored in the field of ocular

drug delivery.6 These dosage forms include liposomes

7-9, nanoparticles

10-13, microemulsions

14-

19 and niosomes

20-21 etc. Oral delivery was considered as a possible noninvasive and patient

preferred route to treat chronic ocular diseases as compared to injectable route. However, re-

stricted accessibility to the targeted ocular tissues limits the utility of oral administration

which demands high dosage to give significant therapeutic efficacy. This can result in system-

ic side effects. Hence, parameters like safety and toxicity need to be considered when expected

to obtain a therapeutic response at the eye site upon oral administration.22

On another side,

topical eye drops, being conventional drug delivery systems are bounded with their own tradi-



tional shortfalls. This shortcoming has led to the evolution of potential use of microemulsions

as an ocular drug delivery carrier as it offers several favorable pharmaceutical and biopharma-

ceutical attributes such as excellent thermodynamic stability, phase transition into liquid-

crystal state, less surface tension, and small droplet size, which may result in improved ocular

drug retention, extended/sustained duration of action and high ocular absorption.23

This research project was undertaken to design ocular microemulsion as colloidal drug deliv-

ery system for increasing solubility of model drug, ebastine and formulating into microemul-

sion based gel for increasing its residential time at the site of action in order to achieve the

therapeutic level of drug for prolong time.

1.1 Definition of Problem

From the literature review on the subject related to ocular delivery and their available thera-

pies, the challenges associated with the current therapies were identified, and from the review

emerged the research problem to be addressed.

A major problem in ocular therapeutics is the attainment of an optimal drug concentration at

the site of action. Poor bioavailability of drugs from ocular dosage forms is mainly due to the

precorneal loss factors which include tear dynamics, non-productive absorption, transient resi-

dence time in the cul-de-sac and relative impermeability of the corneal epithelial membrane. 24

Additionally, most drugs with ocular therapeutic potential have the problem of poor solubility

and hence less bioavailability. To overcome it, various technological strategies are reported in

the literature including micronization, nanosuspension, polymeric micelles and cyclodextrin

based formulation.25

Among various approaches, microemulsions are promising alternative to enhance the ocular

bioavailability of drugs by improved ocular retention, increased corneal/conjunctival drug ab-

sorption and reduced systemic side effects and maintain the simplicity and convenience of the

dosage form as eye drops. Microemulsions are thermodynamically stable, surfactant-

cosurfactant based system, form at low interfacial tension. They are good alternative for oph-

thalmic delivery as it offers the pseudo plastic rheology with increased viscosity after applica-

tion and increased ocular retention and possibility of releasing drug in sustained and controlled

way, increased shelf life, lastly reducing dose and dosing frequency. Microemulsions are also



used to formulate poorly water-soluble drugs since their structure allows solubilization of lip-

ophilic drugs in the oil phase.26-27

Ebastine is a BCS Class II drug. Ebastine is available as an oral antihistamine formula, such as

tablets and syrup, for allergic disorders. But there is no topical formulation. Oral ebastine

causes unfavorable effects on heart like QT prolongation, severe gastric distress, decreased

tear production, resulting in dryness of the ocular surface, which exacerbates ocular discom-

fort and increasing susceptibility of eye to irritation.28,

29

Topical antihistamines are preferred

over oral agents since their direct application at the site of action results in rapid onset and su-

perior efficacy with less systemic side effects. To avoid systemic side effects and ocular dis-

comfort, topical ocular therapy could prove to be superior to systemic therapy in treating ocu-

lar allergies30

. Hence, topical formulation was developed to achieve onsite exposure of ebas-

tine for ocular allergies. Moreover, conjunctiva is more accessible to hydrophilic molecules

than lipophilic molecules. This creates challenge for a lipophilic molecule such as ebastine for

topical ocular development. Successful dissolution of ebastine in o/w microemulsion allows its

use in more convenient soluble form.

Hence, the objective of the present investigation was to design and develop microemulsion

based gel of antiallergic drug ebastine with view to increase the topical bioavailability, im-

prove residence time at eye site and provide sustained delivery of drug for longer period of

time. This formulation offered as a promising strategy for topical drug delivery rather than

systemic drug delivery for ocular allergic manifestation.

1.2 Aim of Research Work

The aim of present work was to design and evaluate the potential of colloidal dispersion sys-

tem ―Microemulsion‖ for ebastine, to treat disorders of the anterior segment of eye via topical

route with following goals,

To prohibit the complications and unfavorable effects of systemic route of drug admin-

istration

To increase bioavailability by topical exposure

Ease of drug application and increased patient compliance



1.3 Objective and Scope of Research Work

This research work was undertaken to design microemulsion based gel of antiallergic drug for

ocular application. The overall objectives of the underlying research work are,

To select the right material, process and optimization design for preparation of Micro-

emulsion

To optimize the formulation of microemulsion with low level of surfactants and the char-

acterization of microemulsion, the characterized parameters need to be eye fitting

To get prolonged drug release, reducing the need for repeated instillation

To target toward affected tissues, reducing possible side effects and required dose

To increase bioavailability of the drug

To prove ocular tolerability and efficacy of said formulation

To estimate the drug concentration in ocular tissue matrix post-instillation of formulation

1.4 Scope of Research Work

In recent years, a dramatically occurrence of ocular diseases reaches to height. Hence, allergic

conjunctivitis demands an urgent need of a novel ocular drug delivery system, which can be

effective and capable of providing sustained therapeutic effect and better patient compliance.

After clinical trials and fulfillment of other regulatory requirements, the developed formula-

tion may prove to be a boon to the society at large for the complete treatment of the allergic

conjunctivitis.

1.5 Rationale of Research Work

1.5.1 Rational for selection of ocular route of drug delivery

Challenges associated with the systemic/oral route31,

32

1. Low and variable oral Bioavailability

The antiallergic drugs including ebastine are reported to have low oral bioavailability be-

cause of the following mentioned reasons

Blood/retinal barrier (BRB)

Blood/aqueous humor barrier (BAB)



Extensive first pass metabolism

Low solubility

High protein binding

Powerful metabolizing enzymes

Variable bioavailability depending on their site of absorption

2. Lack of site specificity

3. Probability of systemic toxicity

4. Requirement of High dose

5. Long term therapy

6. Unfavorable side effect of ebastine like QT prolongation, severe gastric dis-

tress, dryness of eye etc.

To avoid systemic side effects and ocular discomfort, topical ocular therapy could prove to be

superior to systemic therapy in treating ocular allergies

Benefits associated with the topical/ocular route33

The topical application of drug for treatment of ocular diseases are reported to have following

benefits

1) High patient compliance

Self-administrable

Noninvasive

2) Onsite exposure

3) Dose reduction

4) Avoidance systemic toxicity

1.5.2 Rationale for Selection of Microemulsion Drug Delivery System

34,35

Microemulsions are thermodynamically stable, surfactant-cosurfactant based system,

form at low interfacial tension and exhibit high solubilizing potential for hydrophobic

drugs. Successful dissolution of ebastine in microemulsion allows its use in more

convenient soluble form.

Microemulsion are promising alternative to enhance the ocular bioavailability of drugs

by improved ocular retention, increased corneal drug absorption and reduced systemic

side effects and maintain the simplicity and convenience of the dosage form as eye

drops.



Microemulsion show possibility of releasing drug in sustained and controlled way, in-

creased shelf life, lastly reducing dose and dosing frequency.

Microemulsion is good alternative for ophthalmic delivery as it offers the pseudo plas-

tic rheology with increased viscosity after application and increased ocular retention.

The retention period of microemulsion formulation further increased by incorporation

of gelling agent to microemulsion based gel.

1.5.3 Rational for Selection of Drug

Ebastine is the drug which is most benefited if prepared in the form of topical dosage form

because of its variable and less oral bioavailability due to its site specific absorption limitation

and solubility problem. Moreover, ebastine has a high therapeutic index, potency, effective-

ness and differ radically from other existing antihistaminic compounds to give H1 receptor

antagonist action which manages symptoms of early phase of allergy. But, for eradication of

late phase, severe symptoms of allergy, very high drug concentrations are needed and it is un-

likely that this concentration is achieved with oral therapeutic doses. Thus, topical formulation

of said drug could prove to be clinically advantageous.

1.6 Proposed Plan of Work

Ebastine loaded microemulsion and microemulsion based gel were prepared with following

plan of work,

1. Extensive literature survey, selection of drug and excipients, procurement of drug and

excipients.

2. Preformulation study of drug.

3. Formulation development of ebastine loaded microemulsion, screening of microemul-

sion components, development of pseudo ternary phase diagrams using single Smix

system and blend Smix system.

4. Optimization of ebastine loaded microemulsion by applying Design Expert.

5. Characterization of the optimized formulation for globule size, zeta potential, Polydis-

persity index, pH, viscosity, drug content, transmittance, refractive index, osmolarity,

surface tension, electron microscopic studies, in- vitro release study.



6. Formulation development of ebastine loaded microemulsion based Gel.

7. Characterization of microemulsion based gel for appearance, pH, viscosity, drug con-

tent, mucoadhesive strength, spreadability, in- vitro release study.

8. Determination of the in vitro drug release profile of drug loaded microemulsion and

microemulsion based gel.

9. Study of stability profile of drug loaded microemulsion and microemulsion based gel.

10. To perform blinking index and acute irritation study in order to evaluate the in vivo oc-

ular tolerability of formulation.

11. To perform chorioallantoin membrane study in order to evaluate the in vitro ocular irri-

tation potential of formulation.

12. To determine the in vivo performance of formulation by efficacy study using allergic

conjunctivitis animal model.

13. To develop and validate bioanalytical method for determination of drug concentration

in ocular tissue matrix, plasma and measurement of pharmacokinetic parameters.

1.7 Original Contribution by the Thesis

The exclusive work in this research dissertation is authentic. Extensive literature survey was

done to pinpoint the challenges and/or difficulty associated with the complete cure of allergic

conjunctivitis and approaches which overcome them. Although many researchers have been

working on development of microemulsion for various drugs, the idea of the development of

microemulsion of anti-allergic drug by systematic approach of design of experiment for opti-

mization of various parameters which fitted to ocular administration. To head off multiple oral

route-related drawbacks, for treating disease of anterior chamber of eye, has not been investi-

gated till date.

1.8 Outline of Thesis

The overall account on the present research work has been divided into five basic chapters of

the thesis. The introduction, being first chapter includes background, problem definition, aim,

objectives, rationale behind selection of problem, proposed plan of work and origi-

nal/significant contribution by the present research work. The Literature survey, being second

chapter brief about ocular drug delivery, its benefits and challenges, description about disease,



pathophysiology of disease, types of disease, available therapy for treatment, associated chal-

lenges and approaches to overcome them, details of selected dosage form, literature/profile of

model drug and excipients screened for final formulation. The materials and methods, being

third chapter extrapolates as name suggests various materials procured with their resources,

details of equipments used in research work. Beside this, it also includes methodology used in

preformulation studies, analytical and bioanalytical tool, formulation designing with their

evaluation, experimental protocols for in vivo and pharmacokinetic studies. While the results

and discussion, being fourth chapter includes results/ output of experiments performed in pre-

vious chapter and extrapolates inference/significance from them. Lastly fifth chapter conclud-

ed the thesis with summary and discussion.

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drug delivery. Journal of Pharmacy & Pharmaceutical Sciences, 16(5), pp.683-707.

33. Saettone, M.F., 2002. Progress and problems in ophthalmic drug delivery. Business Brief-

ing: Pharmatech, 1, pp.167-71.

34. Chandrakar, S., Roy, A., Choudhury, A., Saha, S., Bahadur, S. and Prasad, P., 2014. Micro-

emulsion: A Versatile Tool for Ocular Drug Delivery. Asian Journal of Pharmacy and

Technology, 4(3), pp.147-150.

35. Vandamme, T.F., 2002. Microemulsions as ocular drug delivery systems: recent develop-

ments and future challenges. Progress in retinal and eye research, 21(1), pp.15-34.


CHAPTER 2 Literature Review

CHAPTER 2

Literature Review



2.1 Overview

Over the last decades, allergic disorders have increased dramatically. Out of different allergic

manifestation, ocular allergy is one of the most common eye disorders in clinical practice.1

with up to 40% of the general population reporting ocular symptoms consistent with allergic

conjunctivitis (AC). In the United States alone, approximately 74 million adults suffer from

AC. Drug delivery in ocular therapeutics is a challenging problem and is considered as an area

of interest to researchers working in the multi-disciplinary areas pertaining to the visionary

organ ―Eye‖, including chemical, biochemical, pharmaceutical, medical, clinical and toxico-

logical sciences. Recently, there has been increased attention focused on following objectives

in this era.2

(a) To tailor newer, effective and safe drug substances for different ocular diseases that are

poorly controlled for conventional formulation like eye drops.

(b) To improve existing ocular dosage forms.

(c) To exploits newer delivery systems for improving the ocular bioavailability of existing

molecules.

(d) To optimized the site specific ocular delivery for attaining site exposure result in dose re-

duction.

Conjunctivitis is inflammation (swelling) of the conjunctiva. The conjunctiva is the transpar-

ent membrane (thin layer of cells) that covers the white part of the eyeball and the inner sur-

faces of the eyelids.3-6

There are three different types of conjunctivitis, each with a different cause. The three types

are:

Irritant conjunctivitis

Infective conjunctivitis

Allergic conjunctivitis



Irritant conjunctivitis

Irritant conjunctivitis occurs when an irritant, such as chlorine (a chemical that is often used to

purify water) or an eyelash, gets into the eyes and makes them sore. Rubbing the eyes can

make the condition worse. The conjunctivitis should settle once the irritant is removed. If the

eyes are very red and painful, medical attention should be sought immediately.7, 8

Infective conjunctivitis

Infective conjunctivitis is caused by a virus, bacteria or a sexually transmitted infection (STI),

such as chlamydia or gonorrhoea. The most common symptoms include reddening and water-

ing of the eyes and a sticky coating on the eyelashes. 7,

8

Allergic conjunctivitis

Allergic conjunctivitis occurs when the eyes come into contact with an allergen. An allergen is

a substance, such as pollen or animal fur, that makes the immune system (the body's defence

system) react abnormally. This causes irritation and inflammation, known as an allergic reac-

tion. Allergic conjunctivitis causes itchy, swollen eyes. 7,

8

FIGURE 2.1 Schematic representation of allergic cascade in Allergic conjunctivitis8



2.2 Allergic Conjunctivitis

Ocular allergy also called allergic conjunctivitis represents a group of hypersensitivity disor-

ders where the eyes produce an abnormal immunological response to normally harmless anti-

gens (allergens) resulting in symptoms such as itching, tearing, burning, foreign-body sensa-

tion and ocular dryness.9 The last four decades have actually seen an exponential increase in

allergic diseases with about 15–20 % of the world‗s population suffering ocular allergies

alone. It is reported that up to 40 % of people of all age groups across the globe are affected by

allergic diseases in its various forms. The estimated cost burden in treating ocular allergy in

the United States alone is approximately 5.9 billion dollars.10

Allergic conjunctivitis affects up

to 40% of the general population. It is also called as ―pink eye." it is an inflammation of the

conjunctiva, the tissue that lines the inside of the eyelid and helps keep the eyelid and eyeball

moist.11

Symptoms usually consist of low-grade ocular and periocular itching (pruritus), red-

ness in the white of the eye or inner eyelid, tearing (epiphora), burning, stinging, photophobia,

watery discharge and swelling of the eyelid. Redness, itching and edema seem to be the most

consistent symptoms of allergic conjunctivitis.12,

13

The conjunctiva can be triggered by allergy

factors like airborne pollens, animal dander, and other environmental antigens.14

2.2.1 Types of Allergic Conjunctivitis

Allergic conjunctivitis involves seasonal allergic conjunctivitis (SAC), perennial allergic con-

junctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratocongiunctivitis (AKC) and

giant papillary conjunctivitis (GPC).15-21

These are caused by different allergens and may have

slightly different symptoms.

Seasonal and perennial allergic conjunctivitis

Seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) are the most

common forms of ocular allergies. These types of allergy are said to affect at least 15–20%

of the population. The presence of specific IgE antibodies to seasonal or perennial aller-

gen can be documented in almost all cases of SAC and PAC.

Seasonal and perennial allergic conjunctivitis are usually caused by

Pollen from grass, trees or flowers

Dust mites



Flakes of dead animal skin

The pathogenesis of allergic conjunctivitis is predominantly an IgE-mediated hypersen-

sitivity reaction. It is caused by an allergen-induced inflammatory response in which al-

lergens interact with IgE bound to sensitized mast cells resulting in the clinical ocular al-

lergic expression. Activation of mast cells induces enhanced tear levels of histamine, tryp-

tase, prostaglandins and leukotrienes. This immediate or early response lasts clinically for 20–

30 min. Mast cell degranulation also induces activation of vascular endothelial cells, which in

turn expresses chemokine‗s and adhesion molecules such as intercellular adhesion molecule

(ICAM), vascular cell adhesion molecule (VCAM). Other chemokines secreted include chem-

okines, monocyte chemo attractant protein (MCP), interleukin (IL)- 8, eotaxin, macrophage

inflammatory protein (MIP)-1 alpha. These factors initiate the recruitment phase of inflamma-

tory cells in the conjunctival mucosa, which leads to the ocular late-phase reaction (Figure 2.2)

FIGURE 2.2 Seasonal and perennial allergic conjunctivitis

Signs and symptoms of these two conditions are the same. The difference is the specific aller-

gens to which the patient is allergic. SAC is usually caused by airborne pollens. Signs and

symptoms usually occur in the spring and summer, and generally abate during the winter

months. PAC can occur throughout the year with exposure to perennial allergens. Diagnostic

features of SAC and PAC consist of itching, redness, and swelling of the conjunctiva.



Vernal keratoconjunctivitis

In this type of allergic conjunctivitis, a nonspecific hyper reactivity occurs that exhibit the

ocular symptoms induced by nonspecific stimuli such as wind, dust and sunlight as well as

their variability, which is not related to allergen levels in the environment.

VKC is a chronic allergic inflammation of the ocular surface mediated mainly by Th2-

lymphocyte. It also have a role in a complex pathogenesis by the over-expressing mast cells,

eosinophil‗s, neutrophils, Th2-derived cytokines, chemokines, adhesion molecules, growth

factors, fibroblast and lymphocytes.

Symptoms include ocular itching, redness, swelling and discharge. Itching may be quite severe

and even incapacitating, if untreated, result in photophobia. The most characteristic sign is gi-

ant papillae on the upper tarsal conjunctiva as shown in Fig. 2.3. The giant papillae is

‗cobblestone-like‗ swellings of several millimeters in diameter.

FIGURE 2.3 Vernal keratoconjunctivitis

Atopic keratoconjunctivitis

Atopic keratoconjunctivitis (AKC) is a bilateral chronic inflammatory disease of the ocular

surface and eyelid. Its pathogenesis mechanism involves both a chronic degranulation of the

mast cell mediated by IgE, and immune mechanisms mediated by Th1- and Th2-lymphocyte

derived cytokines, eosinophils and other inflammatory cells also play a role in its pathogene-

sis. It is considered as ocular counterpart of atopic dermatitis, or atopic eczema. Eczematous



lesions can be found on the eyelids, they are itchy The eyelid skin may be chemotic with a

fine sandpaper-like texture as shown in Fig. 2.4.

FIGURE 2.4 Atopic keratoconjunctivitis

Giant papillary conjunctivitis

Giant papillary conjunctivitis is caused by

Contact lenses

Stitches (sutures) used in eye surgery

Prostheses (artificial) part of the eye that is fitted during eye surgery

GPC is not an allergic disease. Giant papillary conjunctivitis (GPC) is an inflammatory disease

characterized by papillary hypertrophy of the superior tarsal conjunctiva.

Tear samples of patients suffering from GPC do not show increase in IgE or histamine.

While, release of some mediators (CXCL8 and TNF-α) from injured conjunctival epithelial

cells due to mechanical trauma and chronic irritation observed in tear samples. (Fig. 2.5)

FIGURE 2.5 Giant papillary conjunctivitis



2.2.2 Complications of Allergic Conjunctivitis22-24

Allergic conjunctivitis is a condition caused due to the allergy which can lead to lot of site

specific and nonspecific complications in long term. Eventually, allergic conjunctivitis may

be disabling or even sight-threatening. Some of the possible complications are discussed be-

low

Cluster headache: Allergic conjunctivitis dramatically increases the risk of migraine due to

cluster headache.

Keratitis: Cornea (the clear layer at the front of the eye) becomes inflamed (swollen), leading

to the formation of ulcers (open sores).

Iritis: A type of uveitis (inflammation of the middle layer of eye) that causes pain, headache.

Dry eye syndrome: Prolonged untreated allergic conjunctivitis may become cause of second-

ary anterior disease of eye called as dry eye syndrome also known as keratoconjunctivitis sicca

(KCS). It shows similar symptoms like irritation, redness, discharge, and easily fatigued eyes.

Conjunctivitis affect function of goblet cells, eye does not produce enough tears results in dry

eye syndrome.

Complete blindness: Untreated allergic conjunctivitis may lead to permanent blindness.



Oral

Oral antiallergic thera-

peutics reduces symp-

toms of conjunctivitis,

but required therapeu-

tic drug concentration

can‗t be achieved at

site and leads to dry

eye syndrome.28

Injectables

• Injectable delivery

is not well tolerated

by patient.

• Systemic absorp-

tion.28

Topical

• The topical admin-

istration gives ad-

vantage of quicker

onset and better effi-

cacy.

• It also adds site spec-

ificity

• Offer patient com-

pliance.28

FIGURE 2.6 Complications of allergic conjunctivitis

2.2.3 Treatment of Allergic Conjunctivitis

Allergic conjunctivitis can be managed by self-help like avoiding long term use of contact

lenses, avoiding exposure to allergen triggering factors, prohibiting excessive rubbing the af-

fected area etc. However, if this does fail to work, the only way is to take antiallergic medica-

tion and in uncontrolled and/or severe case ocular surgery is required followed by immuno-

therapy.25-27

The various technology platforms available in market for treatment of allergic

conjunctivitis are given in Fig.2.7.

FIGURE 2.7 Technology platforms for ocular therapeutics

The mainstay of the management of ocular allergy involves the use of anti-allergic therapeutic

agents such as ocular decongestant, mast cell stabilizers, antihistamine and multiple action an-

ti-allergic agents known as Novel antihistamines.29,30

The novel antihistamines proved their

dual action, they provide mast cell stabilization to avoid release of allergy mediators as well as

exhibit inhibitory effect on released allergy mediators.31

The model drug selected in research

project ―Ebastine‖ categorized under novel antihistamines.



FIGURE 2.8 Treatment / Medication of allergic conjunctivitis

As per the literature, of all treatment, antihistamines predominantly capture the market for

treatment of allergic conjunctivitis. 32

(Fig.2.9)

FIGURE 2.9 Market scenario for treatment of allergic conjunctivitis

OPHTHALMIC/ORAL ANTIHISTAMINE

OCULAR DECONGESANT TREATMENT

OPHTHALMIC MAST CELL

STABILIZERS

ANTI-INFLAMMATORY



The list of antiallergic drugs used in the treatment of allergic conjunctivitis is given in Table

2.1

TABLE 2.1 List of antiallergic drugs used in the treatment of allergic conjunctivitis

Drug Dosage Availability

Ophthalmic Antihistamines

Alcaftadine

(lastacaft)

One drop once daily 0.25% solution (3mL)

Azelastine (optivar) One drop twice daily 0.05% solution (6 mL)

Bepotastine (bepreve) One drop twice daily 1.5% solution (10 mL)

Emedastine (emadine) One drop up to four times daily 0.05% solution (5 mL)

Epinastine (elestat) One drop twice daily 0.05% solution (5 mL)

Ketotifen One drop twice daily every eight

to 12 hours

0.025% solution (Zadi-

tor/OTC: 5 mL; Ala-

way/OTC: 10 mL

Olopatadine (patanol) One drop twice daily at an inter-

val of six to eight hours

0.1% solution (5 mL)

Olopatadine (pataday) One drop once daily 0.2% solution (2.5 mL)

Ophthalmic Mast Cell Stabilizers

Cromolyn (crolom) One to two drops four to six

times daily

4% solution (10 mL)

Lodoxamide (alomide) One to two drops four times dai-

ly for up to three months


Nedocromil (alocril) One to two drops twice a day 2% solution (5 mL)

Pemirolast (alamast) One to two drops four times dai-

ly


Orally administered 'classic' antihistamines

Astemizole 10mg tab Syrup 2 mg/ml 10 mg OD

Cetrizine 10mg tab, Syrup 0.5 mg/drop 10 mg OD

Ketotifen 1mg tab, Syrup 0.02% soln 1 mg bid

Lortadine 10mg tab, Syrup 0.1% 10 mg OD

Oxatomide 30mg tab, Syrup 2.5% 1 mg/kg BW

Terfenadine 60, 120mg tab, Syrup 0.6% soln 60 mg BID, 120 OD

Others

Ketorolac (acular) One drop four times a day

For cataracts: One drop four

times daily

0.5% solution (3, 5, 10 mL)

Loteprednol (alrex) One drop four times daily (shake

well)

0.2% suspension (5, 10 mL)



Abbreviations used: tab = tablet; soln = solution; OD = once daily; BID = twice daily; BW = body weight

2.3 Microemulsion as Colloidal Drug Delivery for Ocular Therapy

2.3.1 Anatomy & Physiology of Eye as Barriers for Ocular Drug Delivery

As the eye is a very complex and sensitive organ, for development an effective ophthalmic

delivery system, thorough knowledge of the structure/anatomy and physiology of the eye is

crucial. The human eye is an approximately spherical, three-layered structure with a diameter

of 24 mm and a mass of about 7.5 g The anatomical section of the eyeball consist of two parts,

anterior segment consisting of two major salient structures, the cornea and the conjunctiva and

the posterior portion consists of the vitreous body, retina, choroid and back of the sclera.

Accordingly, there are two main purposes of ocular drug applications for treatment of ocular

diseases that emerges on the surface (conjunctivitis, keratitis, etc.) and treatment of ocular dis-

eases that emerges in deeper layers (glaucoma, uveitis, etc.).33

The whole spherical structure is divided into three chambers

Anterior chamber positioned between cornea and iris

Posterior chamber, situated between iris and lens

Vitreous chamber extending from lens back to retina

Cornea and conjunctiva are major epithelial barriers for the topically applied drug molecules.

The conjunctiva is a thin translucent layer situated superficially on the sclera. Tight junctions

are present in the conjunctival epithelium that prevents easy penetration of the molecule



FIGURE 2.10 Schematic representation of visionary organ “Eye”

The intraocular environment consists of two main barriers namely blood–aqueous barriers and

blood–retinal barriers, which make the obstacle for delivery and targeting of ocular therapeutic

effectively. The blood–aqueous barrier is located at the anterior segment of eye. It is com-

posed of the endothelial cells in the uvea, nonpigmented epithelium of the ciliary body, which

specifically includes the iris epithelium and iris vessel endothelium with tight junctions that

control both active and passive transport of hydrophilic drugs from the systemic circulation

into the aqueous humor. Reflex stimulation might enhance the lachrymation up to 100-times

compared with the normal basal tear flow that will result in quick drainage of the topically ap-

plied drugs.34

2.3.2 Conventional Vs Colloidal Drug Delivery Platform

The main problem in ocular therapeutics is the maintenance of effective drug concentration at

the physiological action site for a prolonged time period.35

First choice for ocular therapy is

topical route of administration due to ease of application, convenience and noninvasiveness.

Ocular therapeutics meant for the treatment of either surface or intraocular problems of the eye

available in the market are developed usually as eye drops in solutions or suspensions form.36

Lens Blood–retinal barrier

Ciliary body

Iris Cornea

Aqueous Humor

Vitreous

humor Optic Nerve

Corneal

Epithelium

Conjunctival Epithe- Choroid

Sclera



However, the competent protective barrier mechanism of the eye result in reduced ocular bio-

availability of the therapeutic agents.

As a part of in build defensive mechanism, applied drugs are shortly eliminated from the eye

surface via the process of blinking, baseline and reflex lachrymation, and drainage Precorneal

deficit factors affects bioavailability of a ocular therapeutics. It includes tear dynamics, im-

permeability of the corneal epithelium membranes, momentary residence in the fornix con-

junctiva and nonspecific absorption. Whatever quantity of dose administered, only a minute

quantity of drug (less than 5%) is accessible for its therapeutic activity to the desired eye

site.37

Repeated installation of eye drop is necessary for the maintenance of the required ther-

apeutic drug level in the tear film or at the targeted site, but on contrary side the repeated in-

stallation of highly concentrated solutions may cause ocular surface cellular damage and asso-

ciated toxic side effects.

Hence, in order to conquer these physiological barriers, various advances in formulation strat-

egies have been made for the achieving targeted ocular delivery of drugs. Currently, several

approaches are come into view, including hydro-gels, polymeric micelles, microemulsion,

nanoemulsions, nanosuspensions and lipid-based nanocarriers.38

A series of significant

advantages are presented by lipid-based nanocarriers including microemulsions, liposomes,

nanoparticles, cubosomes and niosomes, as ocular therapeutics, such as improving the bioa-

vailability of poorly soluble drugs, sustaining the release, site specificity and the reduction of

dose and side effects as well.39

2.3.3 Microemulsion

The ―Microemulsion‖ concept was introduced in 1940 by Hoar and Schulman. In 1955, he

subsequently coined the term ―Microemulsion‖. Microemulsion is single optically isotropic

fluid, transparent, thermodynamically stable oil and water system and stabilized by a surfac-

tant usually in conjunction with co-surfactant. The diameter of droplets in microemulsion may

be in the range of 20-200 nm.40

Microemulsions are particularly suitable for ocular drug delivery since these can be formulat-

ed to deal with many challenges including increasing residence time, improving ocular tissue

penetration and sustained release. Moreover, microemulsion can be administered through eye

drops and the small droplet size ensures that there is a negligible problem in visibility on ap-



plication. Furthermore, microemulsions are simple, inexpensive, hence, these are easy to pre-

pare and sterilize also.41

They are stabilized pharmaceutical systems consisting of an oil phase, an aqueous phase and a

combination of surfactant and co-surfactant in appropriate ratios. Microemulsions are trans-

parent systems with low viscosity, exhibiting newtonian flow behavior, low surface tension,

and can incorporate both hydrophilic and hydrophobic drugs simultaneously. The role of sur-

factants is to enhance the drug permeability across the ocular tissue and cornea and serve as

permeation enhancers. Accordingly, microemulsion assures good spreading capability onto the

ocular surface and proper mixing with tears. Therefore, microemulsion can be delivered as eye

drops and will result in better patient compliance.42,

43

Selection of aqueous phase, organic phase and surfactant/co-surfactant systems is the im-

portant parameter for the stability of the microemulsion system and their optimization leads to

a considerable improvement in the drug solubility.

2.3.4 Theories of Microemulsion Formation

There are three major theories of microemulsion formation as discussed below

Interfacial or mixed film theory

This theory implies that the interfacial film is responsible for dissimilar behavior of the aque-

ous and oily segments of the interface.44,

45

Thermodynamic theory

According to this theory, spontaneous formation of microemulsion correlated to the low value

of interfacial tension on account of the interface diffusion of surfactant, as well as to the major

entropy contribution that depends on the homogeneous mixing of one phase in the other phase

in the form of numerous small droplets. 44,

45

Solubilization theory

In this theory, microemulsion are considered as swollen micellar systems, in which water or

oil is solubilized in the ―Reverse Micelle‖ structure to form an one-phase system. 44,

45



2.3.5 Components of microemulsion

Oil phase

The physicochemical parameters of the oil phase / lipid phase must be identified and clearly

understood as they play key role for the existence of microemulsion area and solubilization of

model drug as well. Various kinds of vegetable oils/lipids are available for microemulsion

formation including glycerides, partial glyceride of medium-chain and unsaturated long-chain

fatty acids, and polyalcohol esters of medium-chain fatty acids.46

Oils/ lipids consisting exces-

sive long chain of hydrocarbons are found to be incompatible to penetrate the interfacial film

formed by surfactants/co-surfactant form at interface, as compared with those with short chain

of hydrocarbons, whereas the solubilization capability substantially increases with the increase

in the hydrocarbon chain length. Therefore, small-chain oils/lipids are preferred; however,

medium-chain oils/ lipids are frequently used for ocular microemulsion as they are more suit-

able for the formulation of microemulsion.47

Surfactants

It is important to select the appropriate surfactant to design the stable microemulsion system.

The surfactant should be capable of solubilizing and maintaining a very lower level the inter-

facial tension between oil and aqueous phases. Generally, it is observed that surfactants with

low hyrophilic–lipophilc balance (HLB) used prepare of water-in-oil (w/o) microemulsion

system and high HLB (>12) surfactants are used to prepare oil-in-water (o/w) microemulsion

system. Surfactant having HLB value more than 20 requires additionally co-surfactants to re-

duce efficient hyrophilic–lipophilc balance (HLB) up to a range needed for microemulsion

formation.48

All the experimental trials of phase behavior needed to be carried out at surfac-

tant‗s concentrations higher than the critical micelle concentration (CMC) value of the surfac-

tant(s) under study. Additional important characteristics mandatory to keep in view is no or

very low ocular toxicity and quick biodegradation of the surfactant(s) under study. Both ionic

agents, nonionic agents and their combination type of surfactant can be used. Preferentially,

nonionic surfactants, recognized as ―GRAS‖, owing to their properties like improving solubil-

ization, nonirritating, and ability to sustain precorneal retention with enhanced permeability.

Hence, they find most utility in ocular surfactant based formulation.49



Co-surfactant

Apart from proper selection of oil and surfactant(s), formation of stable microemulsion also

correlated with the flexibility of the interface which can be accomplished by adding co-

surfactant to the formulation. On the basis of the virtual significance of presence of polar

groups, they have ability to change the curvature of the interface. The prime functions of co-

surfactants are to offer very low interfacial tension needed for the development of colloidal

dispersions and maintenance of their thermodynamic stability.

In general, for the stable microemulsion preparation, alcohols with low-molecular-weight and

glycols with chain length ranging from C2 to C10 are considered as co-surfactants. 50

As per lit-

erature, ocular irritation potential is inversely related on the chain length of the alcohols,

whereas some study finding revealed that aliphatic n-alcohols with chain length 3 to 8 were

strong irritants as comparative to ethanol. Ruth et al. in their study compared the efficacy of

alcohols, butanol and ethanol as co-surfactants in microemulsion consisting oil phase (isopro-

pyl myristate), surfactant (egg lecithin) and water. They found that amount of ethanol em-

ployed for the development of microemulsion was higher than that of butanol, and variation in

efficacy was due to difference in the length of the carbon chain.51

Aqueous phase

Water, being universal solvent is mostly used as the aqueous phase in microemulsion prepa-

ration. The aqueous phase pH has a great impact on the phase behavior of the microemulsion,

so it is necessary to maintain the required pH. Generally phosphate buffer pH. 7.4, artificial

trea fluid, purified or sterile water used as aqueous phase in most of ophthalmic microemul-

sion. Numerous excipients such as buffers, antibacterial and isotonic agents are retained by the

aqueous phase. Phase diagrams are influenced by the presence of saline when ionic surfactant

is added and reduces the phase inversion temperature of the nonionic surfactants. The preserv-

atives that are usually used in eye drop formulations cannot be incorporated in microemulsion.

They must not interact with surfactants and result in complexes, nor should they be absorbed

in nanodroplets, which would considerably decrease their antibacterial activity.52



Table 2.2 lists out few components used in microemulsion formulation, having ocular compat-

ibility.

TABLE 2.2 List of components used in ocular microemulsion system.

Oils/lipids

• Ethyl oleate

• Isopropyl myristate

• Isopropyl palmitate

• Oleic acid

• Capric–caprylic triglyceride (Miglyol 80)

• Campul MCM EP

• Octanoic acid

Surfactants

• Pure phospholipids (e.g., soya phosphatidyl choline) and mixed phospholipids, sodium cholate

• Hydroxylated phospholipids/lecithin

• Polyglycerol fatty acid esters

• Polyglycerol polyricinoleate

• Propylene glycol fatty acid esters (e.g., polyoxyethyleneglycerol)

• Triricinoleate, Cremophor EL (Macrogol- 1500 glycerol triricinoleate) monobutyl glycerol

• Labrasol

• Span 20 (sorbitan monolaurate)

• Span 80 (sorbitan mono-oleate)

• Tween 20 (PEG sorbitan monolaurate)

• Tween 80 (PEG sorbitan mono-oleate)

• Propylene glycol PEG 200

Co-surfactants

• Ethanol, propanol and 1-butanol

• 1,2-Propanediol, 1,2-butanediol

• Glycerol, glucitol and PEG



2.4 Literature Survey

The extensive literature survey done and the finding summarized in tables given below.

TABLE 2.3 Recent studies on the microemulsion system for the treatment of glaucoma31-

36

Sr.

No Drug / API Oily Phase SMIX Type Remarks/Finding Author(s)

1.

Timolol maleate

Ethyl oleate

Tween 80,

Span 20

W/O

Sustained drug release

from the ME system

and reduced IOP

Hegde RR,

Bhattacharya SS,

Verma A et al53

2.

Timolol

Isopropyl

myristate

Lecithin,

1-butanol

O/W

Sustained release of

timolol from ME was

found in comparison with timolol solution

Gallarate M, Gas-

co MR, Trotta M

et al.54

3.

Levobuno-

lol Isopropyl myristate

Lecithin,

1-butanol

O/W

LB ME as promising

tool for the ophthalmic

drug delivery system

Gallarate M, Gas-

co MR, Trotta M

et al.55

4.

Pilocarpine

nitrate

Isopropyl

myristate

Macrogol-

1500-

glycerol tri-

ricinoleate,

Lecithin,

PEG 200, propylene glycol

O/W

Extended release and

drug- retarding effect

observed from the

ME system

Haβe A, Keipert S.56

5.

Pilocarpine

hydrochlo-

ride

Ethyl oleate

Sorbitan

laurate, poly-

sorbate 80,

alkanol or al-

kandiol

W/O

The developed sys-

tem was nonirritant

and (precorneal

clearance study) re-

tained the drug for a

longer period than an aqueous solution

Alany RG, Rades

T, Nicoll J et al.57

6.

Pilocarpine

hydrochlo-

ride

Ethyl oleate

Tween 80,

Span 80

W/O

Increased ocular bio-

availability of drug

Chan J, El Ma-

ghraby GM, Craig JP et al.

58

7.

Timolol

maleate

Ethyl bu-

tyrate

Pluronic F127,

sodium capry-

late

O/W

ME-loaded gels

have higher drug-

loading and

transport rates as

compared with con-

trol

Li CC, Abraham-

son M, Kapoor Y

et al.59

IOP: Intraocular pressure; LB: Levobunolol; ME: Microemulsion; O/W: Oil in water; W/O: Wa-

ter in oil.



TABLE 2.4 Recent studies on the microemulsion system for treatment of bacterial kera-

titis37-39

Sr.

No Drug / API

Oily

Phase SMIX Type Remarks/Finding Author(s)

1.

Gatifloxacin

Isopropyl

myristate

Tween80,

Transcutol P

O/W

type

MEs have good sta-

bility, greater corneal

adherence and permeability

Kalam MA, Al-

shamsan A, Aljuf-

fali IA et al.60

2.

Ofloxacin

Oleic acid

Tween 80

and ethanol

W/O

type

Sustained drug re-

lease and improved

preocular residence time

Okur NU, Gokce

EH, Egrilmez S et

al.61

3.

Moxifloxacin

hydrochloride

Isopropyl

myristate

Tween 80,

Span 20

W/O

type

Enhanced ocular

bioavailability,

longer precorneal

residence time and

sustained drug re-

lease from the ME

Bharti SK,

Kesavan K62

ME: Microemulsion; O/W: Oil in water; W/O: Water in oil.

TABLE 2.5 Recent studies on microemulsion system for the treatment of uveitis

40-43

S. No Drug / API Oily

Phase SMIX

Typ

e Remarks/Finding Author(s)

1. Dexamethasone Isopropyl

myristate

Cremophor

EL propyl-

ene glycol

O/W

type Provided good

penetration and pro-

longed drug release

as compared with

conventional dosage form

Fialho SL, da Silva-Cunha

63


myristate Tween 80,

propylene

glycol

O/W

type An improved thera-

peutics effect oc-

curred for the treatment of uveitis

Kesavan K,

Kant S, Singh

PN et al.64


myristate

Poloxamer

407, Brij

98, Tween

80

O/W

type ME prepared by

Poloxamer 407 has

better bioavailabil-

ity than ME prepared by Brij 98

Kesavan K,

Pandit JK,

Kant S.65

4. Prednisolone Ethyl

oleate

Span 20,

Tween

80, etha-

nol

W/O

type Protect the drug

from irradiation ef-

fects and helps in

retaining the chemi-

cal potency of drugs

El Maghraby

GM, Bosela

AA.66

ME: Microemulsion; O/W: Oil in water; W/O: Water in oil.



TABLE 2.6 Recent studies on microemulsion for the ocular delivery of immunosup-

presants44-47

S.No. Drug / API Oily

Phase

SMIX Remarks/Finding Author(s)

1. Sirolimus Triacetin Poloxamer 184,

propylene glycol

Suitable for the immuno-

modulatory treatment of

ocular surface disorders

Buech G, Bertelmann E, Pleyer U et al.

67

2. Tacrolimus Isopropyl

myristate

Tween 80, PEG–

block-

poly(propylene

glycol)–block-

PEG

Prepared MEs were nonirritating, nontoxic and showed higher values of AUC and Cmax leading to reduced systemic side effects

Silva-Cunha A, da Silva GR, de Castro WV et al.

68

3. Everolimus Triacetin Poloxamer 184, propylene glycol

Developed system suitable

for preventing corneal-

graft rejection

Baspinar Y, Bertelmann E, Pleyer U et al.

69

4. Cyclosporine

A

Ethyl

butyrate

Brij-97 Slow and sustained release

of drugs observed from

surfactant or ME-laden

gels

Kapoor Y, Chauhan A

70

ME: Microemulsion.



2.5 Drug and Excipients Profile

2.5.1 Ebastine71-74

Name of drug Ebastine

IUPAC Name 4-(4-benzhydryloxy-1-piperidyl)-1-(4-tert-butylphenyl)butan-1-

one

Description

Ebastine is a second-generation H1 receptor antagonist that is in-

dicated mainly for allergic rhinitis and chronic idiopathic urticar-

ial, allergic conjunctivitis.

Molecular formula C32H39NO2

Molecular Weight 469.658 g/mol

Structural formula

CAS no. 90729-43-4

Log P 7.2

Pka 16.45

Water Solubility 6.47e-05 mg/mL

Protein binding Greater than 95%

Metabolism Hepatic (CYP3A4-mediated)

Mechanism of

action

Inhibit early and late phase allergy trigger

factors. Exhibit blockade of the histamine

receptor

Block the release of anti-IgE-induced prostaglandin D2 (PGD2)

and leukotriene C4/D4, inhibit the release of cytokines.



Marketed product

Ebastine is available in different formulations (tablets, fast dis-

solving tablets and syrup) and commercialized under different

brand names around the world, Ebatin, Ebatin Fast, Ebatrol,

Atmos, Ebet, Ebastel FLAS, Kestine, KestineLIO, KestinLYO,

EstivanLYO, Evastel Z, Ebasten (ACI), etc

2.5.2 Campul MCM EP 75,

76

Name of drug Campul MCM EP

Chemical Name Capmul MCM medium chain mono & diglycerides,

caprylic/capric glycerides.

Structure

Description: Capmul MCM, EP is a mixture of mono acylglycerols, containing

variable quantities of di- and tri-acylglycerols, obtained by direct

esterification of glycerol with caprylic (octanoic) acid Medium

chain length mono (60%) and diglyceride (35%) consisting of

83% w/w caprylic acid (C8) and 17%w/w capric acid (C10).

Product Types Capmul MCM is a mono-diglyceride of medium chain fatty acids

(mainly caprylic and capric). It is an excellent solvent for many or-

ganic compounds. It is also a useful emulsifier for water-oil sys-

tems.

Appearance/ Form Liquid/ Semi-solid

Acid Value 2.5 max

Moisture Karl Fischer 0.5% max.



Alpha Monocaprylate 48% min.

Free Glycerol max. 2.5%

Storage Store in a dry place at 68-77ºF.

Pharmaceutical and

Nutritional Applica-

tions

Carrier (vehicle)

Solubilizer

Emulsifier/ Co-emulsifier

Bioavailability enhancer

Penetration enhancer

2.5.3 Tween 80 77-80

Name of drug Tween 80

Chemical Name Polyoxyethylene 20 sorbitan monooleate; Polyethylene oxide sor-

bitan mono-oleate; Polyoxyethylene sorbitan monooleate; Polyox-

yethylene sorbitan oleate; Sorbitan mono-9- octadecenoate poly

(oxy-1, 2-ethanediyl) derivatives; Sorethytan (20) monooleat

Structure

Appearance/ Form Liquid. (Oily liquid.)

Odour fatty (Slight.)

Colour Clear Amber. Yellow.

pH (1% soln/water) 7 [Neutral.]

Specific gravity at 2o°c

(d20/4)

1.06 - 1.10 (Water = 1)



Storage In an airtight container, protected from light.

Pharmaceutical and


tions

Tween 80 has been widely used in biochemical applications includ-

ing: solubilizing proteins, isolating nuclei from cells in culture,

growing of tubercular bacilli, and emulsifying and dispersing sub-

stances in medicinal and food products. It has little or no activity as

an anti-bacterial agent except it has been shown to have an adverse

effect on the antibacterial effect of methyl paraben and related

compounds. Polysorbates have been reported to be incompatible

with alkalis, heavy metal salts, phenols, and tannic acid. They may

reduce the activity of many preservatives.

2.5.4 Labrasol 81-83

Name of drug Labrasol

Chemical Name Glyceryl Caprylate/Caprate

Product Type Labrasol is composed of a well -defined mixture of mono - di- and

tri-glycerides and mono – and di- fatty acid esters of polyethylene

glycol. It is soluble in ethanol, chloroform, methylene chloride, wa-

ter and insoluble in mineral oils.

Structure

Appearance/ Form oily liquid

Odour faint



Colour (gardner scale) < 2.5

Specific gravity at 2o°c

(d20/4)

1.060 to 1.070

Water content < 1.00 %

Storage Preserve in its original container and prevent exposure to air, light,

heat and moisture

Pharmaceutical and


tions

High HLB non-ionic amphiphilic excipient for pharmaceutical

preparations, used as solubilizing agent and bioavailability enhanc-

er for poorly soluble drugs in oral liquid and capsule formulations,

permeation enhancer in topical preparations, surfactant in

nanoemulsions.

2.5.5 Glycerol 84

Name of drug Glycerol

Chemical Name Croderol; E422; glicerol; glycerine; glycerolum; Glycon G-

100;Kemstrene; Optim; Pricerine; 1,2,3-propanetriol; trihydroxy-

propaneglycerol. Propane-1,2,3-triol

Structure

Product Type Glycerin is a clear, colorless, odorless, viscous, hygroscopic liquid;

ithas a sweet taste, approximately 0.6 times as sweet as sucrose



Appearance/ Form Clear viscous liquid

Odour odorless,

Colour colorless,

Density 1.2656 g/cm3 at 150C;

1.2636 g/cm3 at 200C;

1.2620 g/cm3 at 250C

Hygroscopicity Hygroscopic

Storage Glycerin is hygroscopic. Pure glycerin is not prone to oxidation by

the atmosphere under ordinary storage conditions, but it decom-

poseson heating with the evolution of toxic acrolein. Mixtures of

glycerin with water, ethanol (95%), and propylene glycol are

chemically stable Glycerin may crystallize if stored at low tem-

peratures; the crystals do not melt until warmed to 200C. Glycerin

should be stored in an airtight container, in a cool, dry place.

Pharmaceutical and


tions

In topical pharmaceutical formulations and cosmetics, glycerin is

used primarily for its humectant and emollient properties. Used as

a solvent or cosolvent in creams, emulsions and parenteral formu-

lations. Also used in gels and as anadditivein patch applications. In

oral solutions, used as a solvent, sweetening agent, antimicrobial

preservative, and viscosity-increasing agent. It is also used as a

plasticizer, in film coatings and in production of softgelatin cap-

sules and gelatin suppositories.



2.5.6 Propylene glycol 85

Name of drug Propylene glycol

Chemical Name 1, 2-Dihydroxypropane; 2-hydroxypropanol; methylethylene glycol; methyl glycol; propane-1, 2-diol.

Structure

Appearance/ Form Propylene glycol is a clear, viscous liquid

Odour practically odorless liquid with a sweet

Colour colorless

Density 1.038 g/cm3 at 200C

Storage At cool temperatures, propylene glycol is stable in a well-closed

container, but at high temperatures, in the open, it tends to oxidize,

giving rise to products such as propionaldehyde, lactic acid, pyru-

vic acid and acetic acid. Propylene glycol is chemically stable

when mixed with ethanol (95%),glycerin, or water; aqueous solu-

tions may besterilized by autoclaving.

Pharmaceutical and


tions

Propylene glycol has become widely sed as a solvent, extractant

and preservative in a variety of parenteral and nonparenteral phar-

maceutical formulations. It is a better general solvent than glycerin

and dissolves a wide variety of materials. Used as humectant ~ 15

(%), solvent or cosolvent 5-80 (%) in topicals.



2.5.7 Sodium Hyaluronate 86,

87

Name of drug Sodium Hyaluronate

Chemical Name 3S,4R,6R)-3-[[(2R,4R,5S)-3-acetamido-4-[[(2R,4R,5S)-6-carboxy-

3,4,5-trihydroxyoxan-2-yl]oxymethyl]-5-hydroxy-6-

(hydroxymethyl)oxan-2-yl]methoxymethyl]-6-[[(2R,4R,5S)-3-

acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]methoxy]-

4,5-dihydroxy-5-methyloxane-2-carboxylic acid

Structure

Product Type Hyaluronic acid (HA) is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix.

Appearance/ Form Salt

Odour NA

Water Solubility 46.6 mg/mL

Storage Preserve in its original container and prevent exposure to air, light, heat and moisture



Pharmaceutical and


tions

Hyaluronic acid performs its activities as a tissue lubricant and

hence,. It forms a viscoelastic solution in water which makes it suit-

able for aqueous and vitreous humor in ophthalmic surgery. It is

suggested to provide mechanical protection for ocular tissues and

cell layers due to its high viscosity. The elasticity of the solutions of

hyaluronic acid can assist in the absorption of mechanical stress and

the generation of a protective buffer for the tissues. This viscoelas-

ticity enables maintenance of a deep chamber during surgical ma-

nipulation since the solution does not flow out of the open anterior

chamber.

2.5.8 Carboxy methyl cellulose 87,

88

Name of drug Carboxy methyl cellulose

Chemical Name Carboxymethyl ether, sodium salt, cellulose.

Structure

Appearance/ Form Granular Powder

Odour Odourless.

Colour Almost white colored

Density 0.75 g / cm3

Storage Preserve in its original container and prevent exposure to air, light,

heat and moisture



Pharmaceutical and


tions

Widely used as a viscosity modifier, tablet binder, disintegrant, sta-

bilizing agent and suspending agent for powders either for topical

application or parenteral and oral administration. It is also used in

food products and cosmetics.

2.5.9 Sodium perborate 89-91

Name of drug Sodium perborate

Chemical Name sodium;oxidooxy(oxo)borane;hydrate

Structure

Appearance/ Form Powder

Odour Odorless

Colour White

Density 1.73 g/cm3

Storage Preserved in a stoppered bottle

Pharmaceutical and


tions

Sodium perborate is an oxidative preservative that is used in lubri-

cating eye drops.



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CHAPTER 3 Materials and Methods

CHAPTER 3

Materials and Methods



3.1 Material and Equipment Used

Different materials and equipments used for experimental investigations of the present work

along with their sources have been listed in Table 3.1 and 3.2 respectively.

TABLE 3.1 List of material utilized during research work

Category Material Name Resource / Supplier

Drugs Ebastine Bal Pharma Pvt. Ltd, Banglore

Oils

Campul MCM EP Gattefosse India Pvt. Ltd, Mumbai

Oleic acid Yarrow Chemicals Pvt. Ltd, Mumbai

Isopropyl palmitate Yarrow Chemicals Pvt. Ltd, Mumbai

Arachis oil Yarrow Chemicals Pvt. Ltd, Mumbai

Linseed oil Yarrow Chemicals Pvt. Ltd, Mumbai

Light liquid paraffin Yarrow Chemicals Pvt. Ltd, Mumbai

Labrafac Gattefosse India Pvt. Ltd, Mumbai

Ethyl Oleate Yarrow Chemicals Pvt. Ltd, Mumbai

Surfactants

Labrasol Gattefosse India Pvt. Ltd, Mumbai

Tween 80 S.D. Fine Chemicals Ltd, Mumbai

Tween 20 S.D. Fine Chemicals Ltd, Mumbai

Span 80 S.D. Fine Chemicals. Ltd, Mumbai

Cremophore EL Gattefosse India Pvt. Ltd, Mumbai

Luroglycol FCC Gattefosse India Pvt. Ltd, Mumbai

Co-surfactants

Glyc

Propylene glycol S.D. Fine Chemicals Ltd, Mumbai

Isopropyl alcohol S.D. Fine Chemicals Ltd, Mumbai

Ethanol S.D. Fine Chemicals Ltd, Mumbai

Glycerol S.D. Fine Chemicals Ltd, Mumbai

Butanol S.D. Fine Chemicals Ltd, Mumbai

PEG 400 S.D. Fine Chemicals Ltd, Mumbai



Category Material Name Resource / Supplier

Polymer Carboxy Methyl Cellulose Chemdynes Corporation, Rajkot

Sodium Hyaluronate Chemdynes Corporation, Rajkot

Solvent HPLC grade Acetonitrile S.D. Fine Chemicals Ltd, Mumbai

HPLC grade Methanol S.D. Fine Chemicals Ltd, Mumbai

Other

Chemical/Reagent/ Adju-

vant

Ovalbumin Sigma Aldrich, USA

Aluminium Hydroxide S.D. Fine Chemicals Ltd, Mumbai

Sodium Chloride Allied Chemicals Corporation, Vado-

dara

Sodium Hydroxide Allied Chemicals Corporation, Vado-

dara

Ammonium acetate S.D. Fine Chemicals Ltd, Mumbai

Phenylephrine Hydrochlo-

ride

Sigma Aldrich, USA

Dialysis membrane Type 110 (LA 395) Himedia Labs

Membrane filter 0.22µm Merck India Ltd., India



TABLE 3.2 List of equipment utilized during research work

Instrument Name Manufacturer/Make Model

Digital weighing balance Shimadzu Electronic Balances. BL 220H

Melting point apparatus Veego Inst. Corporation,Mumbai VMP-D

Digital pH meter Elico Digital pH meter LI 615

Magnetic Stirrer Remi service Pvt. Ltd. GAMS 76

Particle size analyser Malvern zetasizer Nano ZS

UV spectrophotometer Shimadzu UV1800 240V

HPLC Water, ALLIENCE 2695

FT-IR spectrophotometer Bruker japan Alpha

Differential Scanning Calorimeter PerkinElmer Pyris 1 DSC

Vortex Mixture Spinix Corporation,Danville CA,USA Spinix

Freeze centrifuge Biolab, Israel BL-35 R

Abbe Refractometer RICO Model RSR-1

Transmittance Electron Microscope

Philips, Holland Technai-20

Cone and plate Viscometer AMETAK Wells-Brookfield KU-3

Tensiometer Kruss Tensiometer Model K12PSS

Autoclave Navyug, India NU-144(C)

Laminar Hood Navyug, India CE-GMP

Homogenizer Heidolph, Germany Silent Crusher M

Osmometer Advanced Instruments Inc., USA Model 3250

3.2 Identification of Drug

The procured drug needs to be identified using preliminary test in order to verify as well as

ensure the purity of drug candidate. These preliminary tests carried out prior to formulation

development. Additionally, identification test considered as official compendia test which

provide an aid for verification of identity of articles as they purported. 1

Identification of drug candidate in underlying research work was carried by its physical ap-

pearance, solubility, melting points. The drug candidate also subjected to Fourier-transform



infrared (FT-IR) spectroscopic study as well as differential scanning calorimetry (DSC) study.

3.2.1 Physical Appearance of Drug

The drug candidate was visually observed for physiochemical properties like color, odour, and

physical state then after compared with the reported same parameters of drug.2

3.2.2 Melting Point

Melting point is prime recognition/identification test for several organic substances.

3 The

melting point of drug candidate was determined by using melting point apparatus. The thin

walled capillary tube which is closed at its one end was filled with small amount of drug sam-

ple and kept in the melting point apparatus. The apparatus was programmed to increase the

temperature gradually. The temperatures range over which drug sample melt was observed

and reported.

3.2.3 Solubility

Solubility test was carried as a test for purity. Quantitative solubility of drug was determined

in water and methanol. For this, 10 mg drug sample was taken in a clean test tube, diluted with

solvent by 0.1 ml increment with continuous shaking.4 The solvent requirement for complete

solubilization of drug sample was noted and the solubility was compared with reported values.

3.2.4 FT-IR Study

Fourier transform infrared (FT-IR) spectroscopy is vital tool for characterization of pharma-

ceutical solids in their solid state.5 The identification of the drug sample was carried using Al-

pha Bruker FTIR spectrophotometer. The sample was prepared by KBr pellets technique. The

pellet was prepared with previously uniform mixer of drug sample and potassium bromide in

ratio of 1:20 in mortar pestle, further the mixture compressed at 20 psi for 10 min using KBr

press. The pellet was kept in sample compartment and scanned at transmission mode in the

region of 4000-400 cm-1

.6 The resultant IR spectrum of drug sample was compared with



standard spectrum of drug.

3.2.5 Differential Scanning Calorimetry

The Differential scanning calorimetry (DSC) study performed to determine thermal behavior

of pure drug ebastine, physical mixture of drug with polymers.7 The study was carried using

PerkinElmer Pyris 1 DSC. The samples were sealed in aluminium pans, heated in a nitrogen

atmosphere and thermograms obtained at heating rate 10.000C/min in the range of 50.00

0C to

300.000C.

3.3 Analytical Method

Adequate analytical method development is of prime concern, when it comes to active ingre-

dient or drug content quantification. Beside this, analytical method also required at different

stages of formulation development like preformulation, evaluation and in vitro-in vivo quanti-

fication. At preformulation stage, it was used for selection of excipients. The suitable analyti-

cal method was not only needed for the in vitro drug release study but also for determination

of active ingredient in biological sample. The available method should be accurate, precise

and convenient while the developed method should be additionally validated for different ana-

lytical parameters.

3.3.1 Estimation of Ebastine using UV Spectrophotometry

For the estimation of ebastine in theformulations, UV spectrophotometric methods were alread

y reported in the literature.8-9

Calibration curves were plotted in methanol and methanolic

phosphate –buffered saline (PBS, pH 7.4, 30% v/v) using UV- 1800 spectrophotometer (Shi-

madzu) at 252 nm. The former used for selection of excipients in formulation design, drug

content determination and the later was used for in- vitro release study of formulation.

3.3.1.1 Calibration Curve of Ebastine in Methanol as a Solvent

The calibration curve of ebastine in methanol as a solvent found application in drug content

determination, excipients selection and stability sample analysis.



Preparation of stock solution

Accurately weighed 10 mg of ebastine was transferred to 100 ml volumetric flask. The drug

sample dissolved and volume was made up with methanol up to the mark in order to obtain

stock solution of 100µg/ml.

Preparation of solution for calibration plot

Aliquots of the stock solution of ebastine in methanol (0.2, 0.4, 0.6, 0.8, 1ml) were accurately

pippeted out into 10 ml volumetric flasks and were diluted with methanol up to mark in order

to obtain final concentration in the range of 2-10 μg /ml. The working λmax of drug sample

was determined by scanning in the range of 200-400 nm. The UV absorption of previously

freshly prepared solutions of calibration plot was noted at λmax. The experimental protocol

carried out in triplicate. Mean value of the absorbance (n=3) verses concentration plotted to

obtain calibration curve using drug free solvent as a blank to avoid solvent effect.

3.3.1.2 Calibration Curve of Ebastine in Methanolic Phosphate Buffered Saline (PBS, pH

7.4, 30% v/v)

Preparation of Phosphate –buffered saline pH 7.410

1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 8.02 g of

sodium chloride and 0.2 gm potassium chloride was dissolved in sufficient water to produce

1000 ml.

Preparation of stock solution

Accurately weighed 10 mg of ebastine was transferred to 100 ml volumetric flask. The drug

sample dissolved and volume was made up with methanolic PBS (pH 7.4, 30% v/v) up to the

mark in order to obtain stock solution of 100µg/ml.



Preparation of solution for calibration plot

Aliquots of the stock solution of ebastine in methanolic phosphate buffer (1, 1.5, 2, 2.5, 3ml)

were accurately pippetted out into 10 ml volumetric flasks and were diluted with methanol up

to mark in order to obtain final concentration in the range of 10-30 μg /ml. The working λmax

of drug sample was determined by scanning in the range of 200-400 nm. The UV absorption

of previously freshly prepared solutions of calibration plot was noted at λmax. The experi-

mental protocol carried out in triplicate. Mean value of the absorbance (n=3) verses concentra-

tion plotted to obtain calibration curve using drug free methanolic PBS (pH 7.4) as a blank to

avoid solvent effect.

3.3.2 High Performance Liquid Chromatography (HPLC) Method Development and

Validation

High performance liquid chromatography (HPLC) method was developed and validated using

HPLC with UV detectors. (Water, ALLIENCE, 2695) for determination of concentration of

ebastine in ocular tissue matrix and plasma for pharmacokinetic/ ocular tissue distribution

studies. Phenylephrine hydrochloride was used as an internal standard (ISTD) in the said

study.

Preparation of Standard solution

Accurately weighed quantity of 100 mg of ebastine was transferred into 100 ml volumetric

flask. Dissolved and diluted up to the mark with methanol. It gave standard stock solution with

strength of 1000 µg/ml of ebastine. From the standard stock solution (1000 µg/ml), 10 ml of

aliquot was pipetted out and transferred into 100 ml volumetric flask and diluted up to mark

with methanol. It gave working stock solution with strength 100µg/ml of ebastine. From the

working stock solution (100 µg/ml), 1 ml of aliquot was pipetted out and transferred into 10

ml volumetric flask and diluted up to mark with methanol. It gave working stock solution hav-

ing strength of 10 µg/ml of ebastine.



Preparation of Internal standard solution

Accurately weighed quantity of 100 mg of Phenylephrine HCl (ISTD) was transferred into

100 ml volumetric flask. Dissolved and diluted up to the mark with methanol. It gave stock

solution with strength of 1000 µg/ml of ISTD. From the standard stock solution (1000 µg/ml),

10 ml of aliquot was pipetted out and transferred into 100 ml volumetric flask and diluted up

to mark with methanol. It gave working stock solution with strength of 100µg/ml of ISTD.

From the working stock solution (100 µg/ml), 0.025 ml of aliquot was pipetted out and trans-

ferred into 10 ml volumetric flask and diluted up to mark with methanol. It gave working

stock solution having strength of 0 . 2 5 µg/ml of ISTD.

Preparation of Quality control samples

Quality control samples like LQC, MQC and HQC were prepared by taking the suitable ali-

quots of from the stock solutions of 100 µg/ml of ebastine and final volume make upto 10 ml

with methanol.

3.3.2.1 Analytical Method Development

Standard solution of ebastine and phenylephrine HCL were scanned in the range of 200-400

nm for determination of detection wavelength in HPLC. Different HPLC trial runs were taken

by varying the columns, mobile phase, mobile phase pH, flow rate at selected λmax for optimi-

zation of analytical method to get properly resolved peaks with suitable and appropriate reten-

tion time.

Optimization of chromatographic conditions

Selection of Internal Standard

Various trials for selection of internal standards like pheniramine maleate, fexofenadine hy-

drochloride, and montelukast etc conducted for development of analytical method for estima-

tion ebastine in tissue matrix but their peaks were not shown or not resolved properly at detec-

tion wavelength of ebastine. Of all trials, phenylephrine HCL was selected as an internal

standard as it shown better resolution with peak symmetry.



Procedure for extraction of drug from ocular tissue matrix

Drug content in ocular tissues was determined after the extraction of the drug from the tissues

by liquid-liquid extraction. An excised ocular tissue subjected to homogenization with acetoni-

trile in 1:5 ratio at 2000 rpm using Silent crusher M homogenizer. The lysed samples were

centrifuged for 10,000 rpm at 40C for 10 min. A 500 µl ocular tissue sample was aliquoted in-

to 5ml eppendorf tube. To it 50µl ISTD was added, required volume of standard solution of

required concentration was spiked. All samples were treated with different protein precipitat-

ing solvents and subjected to vortexing for 2 min and centrifuged at 5000 RPM for 10 min at

4ºC. The supernant was carefully separated, transferred to test tube and injected in to HPLC

system.11-12

Procedure for extraction of drug from plasma

Based on the method developed by Kadam et.al, for extraction of ebastine from rat plasma,

liquid-liquid extraction method was used. A 500 µl aliquot of plasma sample was mixed with

50 µl of ISTD working solution and required concentration standard solution of drugs was

spiked. To this, 1 ml of acetonitrile was added. Resulting solution was mixed on vortex mixer

for 2 min followed by centrifugation at 5000 rpm for 10min at 4ºC. The supernant was careful-

ly separated, transferred to test tube and injected to HPLC system.13-14

Bioanalysis of ebastine by RP-HPLC method in ocular tissue matrix and plasma

The chromatographic separation was achieved on Waters X-Terra Shield, Phenomenex C18

(250 mm x 4.5 mm, 5 µm particle size) equipped with guard column. The mobile phase con-

sisted of Methanol: Acetonitrile: Ammonium acetate buffer (80:10:10), 5.5 pH adjusted with

glacial acetic acid. The HPLC system was operated at a flow rate of 1.2 ml/min in the isocratic

mode and 244nm was used as a detection wavelength. Mobile phase utilized for bioanalytical

method for estimation of ebastine in tissue is tried for plasma. All the peaks were resolved

with all the system suitability parameters in limit hence the same method was validated in

plasma.



3.3.2.2 Analytical Method Validation

The bioanalytical method was validated as per ICH guidelines. The validation parameters in-

clude linearity, precision, accuracy, specificity and robustness. Data obtained were analyzed

statistically.15-16

a. Linearity and Range

The linearity for developed HPLC method was established in the range of 3-400 ng/ml of

ebastine (3, 6, 20, 60,200,300,360,400 ng/ml). The concentration of phenylephrine hydrochlo-

ride taken as internal standard was 1 μg /ml. All the experimental trials were replicated thrice.

The linearity regression analysis of the area ratios (analyte /internal standard) Vs. concentra-

tion curve was obtained. The correlation coefficient value used for verification of the linearity.

b. Precision

Precision was determined by replicate analysis of five determinations of three concentration

levels LQC, MQC and HQC. These QC samples were analyzed against the calibration curve

and obtained concentrations compared with the nominal value. The precision was evaluated as

intraday precision and interday precision. The % coefficient of variation (% CV) was calculat-

ed as per following equation.

Intra-day precision: Five replicates of three different concentrations, (LQC, MQC and HQC)

total 15 determinations were analyzed on a single day.

Inter-day precision: Five replicates of three different concentrations, (LQC, MQC and HQC)

total 15 determinations were analyzed on three consecutive days.

c. Accuracy

Accuracy was determined as intraday and interday analysis of five replicates of three concen-

tration levels of LQC, MQC and HQC. These QC samples were prepared from separately pre-



pared stock solutions. They are analyzed against the calibration curve and mean concentra-

tions compared with the nominal value. % Accuracy was calculated as per following equation.

Intra-day precision: Five replicates of three different concentrations, (LQC, MQC and HQC)

total 15 determinations were analyzed on a single day.

Inter-day precision: Five replicates of three different concentrations, (LQC, MQC and HQC)

total 15 determinations were analyzed on three consecutive days.

d. Sensitivity

Sensitivity was carried out by injecting repeatedly Lower Limit of Quantification (LLOQ)

which is the lowest concentration of the standard curve and that can be measured with ac-

ceptable accuracy and precision. The 6 replicates of extracted LLOQ samples (mixture of

0.003 and 0.025µg/ml of ebastine and phenylephrine respectively) were injected repeatedly at

the same chromatographic condition and measurements of retention time, peak area, mean and

%CV were calculated.

e. Extraction Recovery

Recoveries of ebastine and internal standard were determined at 3 concentration levels, HQC,

MQC and LQC. % Recovery of analyte and internal standard were determined after pro-

cessing and inject 6 replicates of QC samples (Extracted samples) and unextracted at same

chromatographic condition. The % recovery was calculated by comparing extracted and unex-

tracted area response of analyte and internal standard as per following equation.

% Recovery = Mean of area ratio of extracted sample × 100

Mean of area ratio of post extracted spiked sample

f. Robustness

The robustness of the analytical procedure was measured from its capacity to remain unaffect-

ed by small but deliberate variations in method parameters like wavelength change (+ 2)



which provided an indication of its reliability during its normal usage.

g. Stability

Short term stability, freeze thaw stability, long term stability and stock solution stability were

performed as per USFDA Guidelines. Short term stability of drugs was determined by com-

paring the mean area ratio of fresh solutions of five replicates of LQC and HQC and stability

samples after 6 hours. Dilutions were stored at room temperature. Stability solutions were in-

jected and analyze against fresh QC samples. For Freeze thaw stability, five replicates of

LQC and HQC were stored in deep freezer for 24 hours and after that thawed it unassisted at

bench top (FT Cycle -1). Refreeze the samples after complete thawing. Withdraw these sam-

ples after a period of minimum of 12 hours and thawed (FT Cycle-2) Refreeze the samples

after complete thawing. Again withdraw these samples after a period of minimum of 12 hours

and after thawing (FT Cycle-3) the samples were analyzed. QC samples were processed and

analyzed, along with freshly prepared calibration curve standards.

Long-term stability is performed to assess the stability of analyte in biological fluids during its

storage in deep freezer below -50°C. It was determined by comparing the mean concentration

of five replicates of LQC and HQC from the freshly prepared calibration curve and stability

samples those that were kept in deep freezer. The stability of stock solutions of drug and the

internal standard should be evaluated for at least 6 hours at room temperature and at refriger-

ated condition (2-8˚C) for 7 days.

h. System suitability

System suitability is the test for checking the system to ensure system performance prior to or

during the analysis of unknown sample. For system suitability test, six replicates of drug sam-

ples were run and repeatability with the parameters like theoretical plate count, tailing factors,

resolution and reproducibility in retention time (RT) were determined. The % RSD was calcu-

lated.

i Statistical Analysis

The experimental data obtained were statistically assessed with the aid of Microsoft Excel.

Linearity was checked by determination of standard deviation, correlation coefficient and line-



ar regression equation. Moreover, accuracy of the developed analytical method was assessed

by % recovery method, standard deviation (SD) and relative standard deviation (% RSD) and

confirmed the precision, robustness, etc. Intraday and interday precision was statistically ana-

lyzed by utilizing ANOVA from Analysis Toolpak, Microsoft Excel. Signal-to-noise ratios

were determined for determination of LOD and LOQ.

3.4 Screening of Components of Microemulsion Formulation17

The major components for microemulsion system include drug, surfactants, and cosurfactants.

Followed by identification of drug, various formulation materials were screened for their suit-

ability for proposed system. The screening of these materials based on maximum solubility of

the drug. Beside, safety profile, compatibility and approval status were also taken into consid-

eration.

3.4.1 Selection of Oil

The solubility of ebastine in different oils was determined by adding excess amount of the

drug to 1 ml of oil in separate stopper glass vials and mixing by vortex mixture. The resultant

mixture was then shaken for 24 hr. in an orbital shaker at 37±20C to reach equilibrium. The

equilibrated samples were removed from the shaker and subjected to centrifugation for 20 min

at 3000 RPM. The aliquots of supernatant were filtered through 0.45 μm membrane filters and

the solubility of ebastine was determined by analyzing the filtrate spectrophotometrically

(Shimadzu 1800, Japan) after dilution with methanol at 252 nm. Appropriately diluted solu-

tions of oils in methanol were taken as blank.

3.4.2 Selection of Surfactants

The selection of surfactants mainly depends on the HLB value, non irritancy and compatibility

of surfactants with each other and oil. The solubility of ebastine in different surfactants was

determined by adding excess amount of the drug to 1 ml of surfactants in separate stopper

glass vials and mixing by vortex mixture. The resultant mixture was then shaken for 24 hr. in

an orbital shaker at 37±20C to reach equilibrium. The equilibrated samples were removed from

the shaker and subjected to centrifugation for 20 min at 3000 RPM. The aliquots of superna-



tant were filtered through 0.45 μm membrane filters and the solubility of ebastine was deter-

mined by analyzing the filtrate spectrophotometrically (Shimadzu 1800, Japan) after dilution

with methanol at 252 nm. Appropriately diluted solutions of surfactants in methanol were tak-

en as blank.

3.4.3 Selection of Co-surfactants

Selection of co-surfactant is based on their capability to form the stable micro-emulsion with

the selected surfactant at minimum concentration. The solubility of ebastine in different co

surfactants was determined by adding excess amount of the drug to 1 ml of co surfactants in

separate stopper glass vials and mixing by vortex mixture. The resultant mixture was then

shaken for 24 hr. in an orbital shaker at 37±20C to reach equilibrium. The equilibrated samples

were removed from the shaker and subjected to centrifugation for 20 min at 3000 RPM. The

aliquots of supernatant were filtered through 0.45 μm membrane filters and the solubility of

ebastine was determined by analyzing the filtrate spectrophotometrically (Shimadzu 1800, Ja-

pan) after dilution with methanol at 252 nm. Appropriately diluted solutions of co-surfactants

in methanol were taken as blank.

3.4.4 Drug Excipients Compatibility Study

Drug excipients compatibility study was performed to ensure the physical and chemical com-

patibility of components of formulation. The screened components of formulation surfactants,

cosurfactants were considered for further development only if physically and chemically com-

patibility with drug was observed.

FT-IR spectra of pure drug , drug with oil/surfactants/co surfactants, drug with polymers

stored at 25 ± 2 0 C, 60% ±5% relative humidity for a period of 7 days were recorded in the

range of 4000-400 cm-1

using FT-IR Spectrophotomer (Bruker Alpha-one, Bruker Optik,

Germany).18-19

A FT-IR spectrum gives the identification of specific functional group and so

that from this any chemical incompatibility if occurred can be easily identified by change or

shift in the peak of specific functional groups of drug.

3.5 Construction of Pseudo- Ternary Phase Diagrams

Pseudo ternary phase diagrams were used to determine the existence of microemulsion region.



After selection of appropriate microemulsion components, the pseudo ternary phase diagrams

were constructed to identify the nature of microemulsion regions at ambient temperature

(25°C). Large number of batches of different composition must be prepared to construct the

phase diagram. Pseudo-ternary phase diagrams were constructed using previously screened

components by Prosim software.20

Various phase diagrams were prepared with weight ratios

of surfactant to cosurfactant (Smix) individual and blend system. Individual Smix system con-

sist of Labrasol and Propylene glycol while blend Smix system consist of mixture of two sur-

factants viz. Labrasol and Tween 80 as well as mixture of two co- surfactants viz. Propylene

glycol and Glycerol. Double distilled water was used as an aqueous phase. The selected indi-

vidual Smix ratio (2:1) was further studied by blend Smix ratios 2(1:1):1, using mixtures of

surfactants and 2(1:1): 1(1:1), using mixtures of surfactants and co-surfactants. The Smix

blend ratio which produced broader microemulsion region was selected for formulation opti-

mization. This attempt was made to keep the surfactants concentration as low as possible in

the ophthalmic formulation to avoid any associated toxicity and ensure eye fitting. A blend of

surfactants with an HLB that matches that of the oil phase will provide better solubilization

and stability of the dispersion system produced.21

For each phase diagram at a specific surfac-

tant/cosurfactant weight ratio, the ratios of oil to the mixture of surfactant and cosurfactant

were varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. The mixtures of oil, surfactant and

cosurfactant at certain weight ratios were diluted with water drop wise, under moderate mag-

netic stirring. After being equilibrated, the mixtures were assessed visually and determined as

being microemulsion, crude emulsions or gels. Gels were defined for those clear and highly

viscous mixtures that failed to show any change in the meniscus after tilted to an angle of 90°.

The concentration of water at which transparency to turbidity transition occurred was derived

from the weight measurement. These obtained values further used to determine the boundaries

of the microemulsion domain corresponding to the value of oil, as well as surfactants and co

surfactants ratio.

3.6 Preparation of Drug Loaded Microemulsion

Based on the solubility data, Capmul MCM EP, Tween 80 and Labrasol, Propylene Glycol

and Glycerol were selected as oil, surfactant blend and co-surfactant blend respectively. The

microemulsion was prepared by water titration method. The following scheme was used to



Drug dissolved in an appropriate quantity of oil phase

In this mixture, add weighed quantity of mixture of surfactant and co-surfactant (Smix)

Double distilled water was added drop by drop to the oil and Smix mixture under magnetic stirring at ambient temperature.

Transparent and clear microemulsion was taken as the end point of aqueous titration method.

prepared microemulsion,

3.7 Optimization of Microemulsion by D-Optimal Mixture Design

D-optimal mixture design (Design-Expert 7.0.0 (Stat-Ease Inc., Minneapolis, USA) was se-

lected because the generalized variance of the estimates of the coefficients is minimized.22

The

software selected a set of candidate points as a base design included factorial points (high and

low level from the constraints on each factor, centers of edges, constraint plane centroids, axi-

al checkpoint, and an overall center point).

It is commonly used to reveal main effects and interaction effects between the independent

variables of the experiment. Moreover, the numbers of trials required are less. Twelve runs

were carried out to optimize microemulsion formulation. Different design constraints, i.e. A

(amount of oil), B (amount of Smix), and C (amount of water) were taken at high and low lev-

els. The sum of A, B, and C were kept fixed at 100%. The effect of these formulation variables

was studied on the % Transmittance, Globule size and Viscosity. Validity of experimental de-

sign was confirmed by plotting a standard error of design graph. The probability value (α) for

determination of statistical significance was set at 0.05, which indicated that a ―hypothesis‖

theory would be rejected if their corresponding p-values were ≤ 0.05.23

Models were selected

on the basis of sequential comparison and lack of fit test. Significance of the models was fur-

ther confirmed by statistical analysis. Response surface, contour plot, residual plot and overlay

plots were constructed for the response variables.



3.7.1 Preparation of Drug Loaded Microemulsion

The D-optimal design suggested different combinations of oil, Smix, and water. The suggested

quantity of oil and Smix was mixed using a magnetic stirrer to produce the oily phase, at this

stage the ebastine was dissolved in the oily phase. Finally aqueous phase was added drop wise

to obtain drug loaded microemulsion formulation.

3.8 Formulation and Optimization of Microemulsion Based Gel

The optimized microemulsion has very low viscosity, which may restrict its topical applica-

tion. To overcome this, gelling agents were incorporated into formulation. The ocular delivery

improved by adding mucoadhesive polymer in previously made microemulsion formulation.

The weight ratio of Carboxy methyl cellulose (CMC 1%) and Sodium Hyaluronate (SH 1.5%)

was found satisfactory based on proper gel formation. The former polymer used in commercial

ocular formulations, as it has desirable mucoadhesive and a high retention time on the ocular

surface and the latter one exhibit excellent viscoelastic, lubricating and water retention proper-

ties. The literature revealed that, this combination benefited with high viscosity under low fric-

tion conditions (between blinking) which stabilizes the tear film and low viscosity under high

friction conditions (during the blinking) which reduces discomfort in animal as well as hu-

mans.24

The microemulsion formulation made as per previously optimized formulation and processing

constraints were subjected to sterilization by membrane filter. Then after, polymer dispersion

was formed by suspending polymers in water. The polymer dispersion kept for overnight to

form viscous gel matrix. Prepared microemulsion and polymer dispersion was mixed in 1:1

v/w ratio.25

Smooth viscous, transparent gel was formed. The underlying procedure for prepa-

ration of microemulsion based gel was carried out strictly in aseptic area to maintain the steril-

ity of overall formulation.



3.9 Evaluation of Optimized Microemulsion Formulation

3.9.1 Measurement of pH

The pH of the ocular formulation is very important for avoiding the irritation to ocular tissue.

For optimized formulation, pH was measured using pH meter (Li 615, Elico, India, Hydera-

bad) which was previously calibrated using standard buffers of pH 4 and pH 7 as per the es-

tablished procedure.26-

27

3.9.2 Droplet size, Zeta Potential and Viscosity Measurement

The droplet size of the microemulsion was determined by photon correlation spectroscopy

(which analyzes the fluctuations in light scattering due to the Brownian motion of the parti-

cles) using a Malvern zeta sizer (Nano ZS, Malvern instruments, UK), Zeta sizer able to

measure sizes between 10 and 5000 nm. The measurements were performed at 25 °C at a 90 °

angle. 28

Each size value reported was the average of at least three independent measurements.

Samples were suitably diluted with double distilled filtered water to avoid multi-scattering

phenomena and then placed in quartz cuvettes. The real and imaginary refractive indexes were

set at 1.59 and 0.0, respectively. Zeta Potential was determined by Zeta sizer (Malvern instru-

ments UK) using clear disposable zeta cell and filed strength of 20 V/cm was employed. The

electrophoretic mobility was converted into to the zeta potential. The viscosity of microemul-

sion was determined by Ostwald type capillary viscometer at room temperature.29

3.9.3 Measurement of Refractive Index

After application of eye drops, possible impairments of vision or discomfort to the patient is

detected by refractive index measurements. Refractive index indicates the isotropic behavior

of formulation and also proved the transparency of formulation. The refractive index of the

system was measured by Abbe Refractometer (RICO, Model RSR-1) by placing one drop of

the formulation on the slide in triplicate at 250C and compared it with water.

30-32



3.9.4 Measurement of Osmolarity

Evaluation of osmolarity using an osmometer is of vital importance for physiological ac-

ceptance of the formulation by ocular tissues. Osmolarity of optimized formulation measured

using Osmometer (Advanced Instruments Inc., USA; Model 3250).32-33

3.9.5 Measurement of Surface Tension

Surface tension determination ensures the uniform spreading of the formulation on the corneal

and conjunctival surface. Surface tension of optimized formulation measured using Tensiome-

ter (Kruss Tensiometer; Dimensions: 19.900 mm*0.200mm*10.00mm, Model K12PSS).32,

34

3.9.6 Determination of Drug Content

The drug content of the optimized microemulsion formulation was determined by extracting

0.5 ml formulation containing drug equivalent to 5 mg with methanol. After suitable dilutions

with methanol, absorbance was determined using UV spectrophotometer35

(Shimadzu UV-

1800, Japan) at 252 nm.

3.9.7 Transmission Electron Microscopy

To study the microstructures of microemulsion, transmission electron microscopy is the most

important technique as it directly produces high-resolution images. It can capture any co-

existent structure and microstructural transitions36

. The morphology of formulation was per-

formed using TEM (Technai-20, Phillips, Holland, Electron source: LaB6, Tungsten Fila-

ment). A drop of sample was placed onto a carbon coated grid on a single tilt sample holder to

form a thin liquid film. The excess solution was removed followed by negative staining with

1% phophotungstic acid. The sample was examined and simultaneously photographed at an

accelerating voltage with point resolution 0.27nm and magnification up to 25x to 7, 50,000x.



3.9.8 Measurement of % Transmittance

The microemulsion formulation was diluted (10 times dilution) with distilled water. Thereaf-

ter, transparency of formulation was determined at 650 nm with purified water taken as blank

using UV spectrophotometer.37

3.10 Evaluation of Microemulsion Based Gel

The formulated microemulsion based gel evaluated for physical examination like pH, rheolog-

ical assessment, mucoadhesive strength, drug content, spreadability and in vitro release study.

3.10.1 Measurement of pH

As discussed previously, pH of the ocular formulation is very important for avoiding the irrita-

tion to ocular tissue. The pH of 1% w/v aqueous solution of the prepared microemulsion based

gel was measured using pH meter (Li 615, Elico, India, Hyderabad) which was previously cal-

ibrated using standard buffers of pH 4 and pH 7 as per the established procedure.

3.10.2 Rheology Study

Viscosity is most important parameter for topical formulation.

38 The viscosity of the formula-

tion should be such that it not only improves the therapeutic performance of formulation but

also convenient during instillation at desired site. The rheograms of microemulsion gel and

microemulsion based gel diluted with tear fluid in a ratio of 40:7 were determined at different

shear stress using Plane and cone viscometer.39

3.10.3 Mucoadhesive Strength

The mucoadhesive force was determined using modified two-pan balance method.40

As per

the literature review, in spite of numerous studies concerning the in-vitro and in-vivo perfor-

mance of mucoadhesive drug delivery systems, surprisingly, there has not been any standard

technique designed for mucoadhesive measurement or any analytical method that can be em-

ployed to qualify mucoadhesive strength. In-vitro tests including two-pan balance method are

the most common and convenient methods to assess the mucoadhesive properties of formula-



tions.41

In this method, one side of the balance was provided with blocks at the top for balanc-

ing and the other side had a receptacle for water. 20 µl gel test sample was applied on the per-

fectly horizontal surface and was just touched with the cellophane membrane (1 cm2) sticked

at the horizontal end opposite to that of water receptacle. Gradually water was added drop by

drop till the cellophane membrane got detached from the gel. Weight in grams of water re-

quired to separate the two surfaces was measured and mucoadhesive force was calculated us-

ing following equation,

F = w x g

Where F is the mucoadhesion force (dynes / cm2),

w is the minimum weight required to break the bond (grams), g is the

acceleration due to gravity (cm/s2).

3.10.4 Spreadability

Spreadability of the gel was determined by taking 0.5 g gel between two cellophane mem-

branes and placing 100 g weight on it for 1 minute to make simulated blinking response.42

The

diameter of the area in which the gel got spread was measured.

3.10.5 Drug Content

The drug content of the microemulsion based gel formulation was determined by extracting

formulation containing drug equivalent to 10 mg with methanol. After suitable dilutions with

methanol, absorbance was determined using UV spectrophotometer43

(Shimadzu UV-1800,

Japan) at 252 nm.

3.10.6 In vitro Drug Release Study

In-vitro drug release profile of the microemulsion and microemulsion based gel were studied

by dialysis bag/ dialysis sac technique.44,

45

In this method, microemulsion and microemulsion based gel were filled in the activated dialy-

sis bag (Dialysis membrane 110, LA 395, Himedia, 12 kDa) and suspended in a glass beaker

containing 50 ml of methanolic phosphate buffered saline (pH 7.4, 30% v/v).46

The system



was maintained at 32 ± 0.50C to mimic physiological condition of eye surface temperature

with continuous stirring on magnetic stirrer at 150 rpm. The system was covered with paraffin

film during experiment to prevent any evaporative loss.47

At periodic time interval, the ali-

quots were withdrawn from the receptor compartment for 24 hrs. The samples were analyzed

spectrophotometrically at 254 nm. Sink condition was maintained in the receptor compartment

during in vitro release studies. Each sample analysis was performed in triplicate.

3.10.7 Kinetics of Drug Release Study

To study the release kinetics of microemulsion and microemulsion based gel formulation, re-

lease data fitted to the following equations 48-50

3.10.7.1 Zero Order Release Equation

Qt = k0.t

Where Qt is the percentage of drug released at time t and k0 is the zero order release rate con-

stant.

The Regression coefficient (R2) value of plot of amount of drug released (Qt) verses time (t),

nearer to unity indicates zero order, concentration independent release pattern.

3.10.7.2 First Order Release Equation

ln (100-Qt) = ln 100 – k1.t

Where Qt is the percentage of drug released at time t and k1 is the first order release rate con-

stant.

The Regression coefficient (R2) value of plot of log cumulative % drug remaining verses time,

nearer to unity indicates first order, concentration dependent release pattern.

3.10.7.3 Higuchi Square Root of Time Model

Qt = kh.t1/2

Where Qt is the percentage of drug released at time t and kh is the Higuchi square root of time

release rate constant.

The regression coefficient (R2) value of plot of percentage drug release verses square root of

time nearer to unity indicates Fickian diffusional release mechanism.



3.10.7.4 Korsmeyer-Peppas Model

Qt/Q∞= kKP . tn

Where Q∞ is the total drug released after infinity, Qt/Q∞ is the fraction of drug released at time

t, kKP a constant compromising the structural and geometric characteristics of the device, and

n, the release exponent, which is indicative of the mechanism of drug release.

A plot of log (Qt/ Q∞) versus log t gives straight line of gradient n and an intercept of log K.

Values of exponent n and the corresponding release mechanism are given in Table 3.3

TABLE 3.3. Values of diffusional exponent and corresponding release mechanism

Diffusional Exponent, n Type of Transport (release ) Time Dependence

n=0.5

0.5<n<1

n = 1

n>1

Fickian diffusion

Anomalous transport

Case II transport

Super case II transport

t1/2

tn-1

time independent

tn-1



3.11 Sterilization and Sterility Testing

3.11.1 Sterilization

The optimized microemulsion formulation was subjected to sterilization using membrane fil-

tration unit by passing it through 0.22 µm membrane filter maintaining aseptic condition.51

3.11.2 Sterility Testing

Sterility testing of microemulsion formulation was performed by direct inoculation method to

examine growth of bacteria and fungi. The media used to detect aerobic and anaerobic bacteria

was fluid thioglycolate medium and the media used to detect fungal organism was soyabean

casein digest medium as per IP standards. Three test tubes were taken for negative control,

positive control and sample testing for both medium. For negative control, both sterilized (au-

toclaving at 1210C, 15 psi) medias were taken separately, while for positive control, aerobic

bacteria Staphylococcus aureus and fungal organism Candida Albicans were inoculated in flu-

id thioglycolate media and soyabean casein digest media respectively.52

With the help of ster-

ile syringe, 1 ml of sample was transferred aseptically to both media separately. The samples

were incubated for at 35 ± 10C and 25 ± 1

0C for FTM and SCDM media for 14 days and

checked for turbidity on everyday which is indicate the of microbial growth. As discussed pre-

viously, the gelling agent incorporated into microemulsion system in aseptic cabinet in to

avoid further possible contamination.

3.12 In vitro / In vivo studies

The experimental protocol was approved by the Institutional Animal Ethics Committee

(IAEC) Reference No. 984/01/2017-07 for the use of animal in the study. Utmost care was

taken to ensure that animals were treated in the most human and ethically acceptable manner.

The ocular potential of optimized ocular formulation was assessed by performing in vitro

study like hen's egg chorioallantoic membrane test (HET-CAM), blinking index for tolerabil-

ity, acute ocular irritation study and in vivo antiallergic efficacy study in ovalbumin (OA)-

induced allergic conjunctivitis (AC) in guinea pig model followed by histopathology.



3.12.1 Ocular Irritation Study by Hen’s Egg Chorioallantoic Membrane (HET-CAM)

Test

Selection of Eggs

The eggs were collected less than 1 week after lying and incubated for about 9 days; on 10th

day, their blunt ends are tested by the candling lamp. Only the eggs with emergent vascular

system were selected for the test.

Preparation of the Eggs for Test

Candling procedure helps in identifying the air space and it was marked on the eggs. Then,

after wiping with 70% IPA and a small window was made on the shell at the pointed end of

the egg. The shells of the egg are opened at that marked portion on the blunt ends. The under-

lying membrane was carefully removed in such a way that underlying blood vessels are not

damaged. Exposed chorioallantoic membrane (CAM) was treated with 10 μL of the optimized

formulation. The chorioallantoic membrane was also treated with 10 μL of 1 N NaOH and

considered as positive control and 10 μL of 0.9% w/v NaCl as negative control. The effects

were observed near the surroundings of the applied sample within 5 min. After 5 min, change

in CAM was observed for parameters like hemorrhage, coagulation and lysis.53-55

3.12.2 Ocular Tolerability Study by Blinking index

Testing Protocol for Blinking Index (B.I.)

The animals were held on top of a lab table with a thick absorbent paper. Blinking counts were

performed with an electronic count-up timer over a 5-minute period. Using an adjustable vol-

ume digital pipette, saline and optimized formulation were applied to the lower cul-de-sac

while pulling the upper eyelid gently and tilting the head of the animal slightly, making sure

that the formulation did not spill out before the first blink. A volume of 25 μL was used here

as a substantial stimulus for blinking. The right and left eyes were tested with saline, and the

test solution was tested 30-60 minutes later.56



Mathematical Expression

The ratio between the number of blinks, counted over a 5-minute period following the instilla-

tion of a test solution, and the corresponding number of blinks, counted over a 5-minute peri-

od, in the same animal following the instillation of a normal saline solution, gives the Blinking

Index (B.I.)

The average result obtained consecutively from both eyes of the same animal was entered as a

single value. The results are presented as Mean ± Standard Error of the Mean (S.E.M.).

3.12.3 Acute Ocular Irritation Study

The animals were held in position same as that of previously mentioned blinking index meas-

urement protocol. The saline and optimized formulation were instilled to the lower cul-de-sac

while pulling the upper eyelid gently and the animals were observed up to 60 min for redness,

swelling, watering of the eye.57

3.12.4 Efficacy Study by Ovalbumin Induced Allergic Conjunctivitis Model

Testing Protocol for Ovalbumin-Induced Allergic Conjunctivitis

Animals were sensitized on day 1, 7, 14, and 21 by intraperitoneal injection of ovalbumin (100

µg/0.5 ml/animal), suspended in aluminium hydroxide gel as an adjuvant. Non-sensitized an-

imals used for the experiment received only aluminium hydroxide gel. Seven days after the

last sensitization, animals were used for the experiments assessing efficacy of optimized for-

mulation.58-59

At the time of experiment, a 20 µL optimized formulation, saline, were instilled into the right

eye of respective group using a micropipette and for oral, ebastine (3mg/kg) in 0.5% CMC. At

0.5 and 24 hr. after the instillations, the eye was challenged with ovalbumin solution (100

mg/ml, 30 µl).



3.12.4.1 Edema Scoring

Edema was scored at 15, 30, 60, 90, and 120 min after the instillation of ovalbumin. For eval-

uation of edema, scoring system used.60

The edema scoring was done according to graded scale. Following system was used for as-

signing the edema scores

0- No edema

1- Slight edema

2- Partial eversion of eye

3-Eyelid half-closed

4-Eye swelling, more than half eyelid closed

3.12.4.2 Scratching Behavior

Eye scratching behavior was defined as fore-limb movements over two times directed to the

ocular surface.61

In the same sensitization protocol as describe in 3.12.4. Along with edema,

the number of eye scratches was counted for 30 min. The scratching response was assessed

after topical antigen challenge at 0.5hr.62

3.12.5 Histopathological Study

The eyeballs together with the conjunctiva and lids of animals from the saline, saline + oval-

bumin and optimized formulation group were exenterated and fixed in 10% buffered formalin.

Tissues were subsequently processed for dehydration in a series of ascending alcohol concen-

trations. The samples embedded into paraffin wax and stained with hematoxylin and eosin.63-64

DPX was used as mounting medium and micro toming was performed using microtome (mod-

el 0126, Yorco, India). The histopathological examinations for determination of dam-

age/irritation due to the formulation were performed using inverted microscope. (Nikon TS-

100)



3.13 Pharmacokinetic Study

Pharmacokinetic parameters give valuable information of formulation and can serve as an im-

portant tool in an establishment of IVIVC, which is an important quality attribute.

From a high level perspective, ocular PK studies help to elucidate ADME of formulation. It gives

information about absorption into, distribution and metabolism within and elimination from the

eye (ADME). More precise objectives for these studies typically center on determining ability of

drug to penetrate the target tissue, and determine its concentration, by a specified dosing method.65

Single dose pharmacokinetic study was performed by measuring drug concentration in ocular tis-

sues. For reliable data quality, sampling time should be chosen carefully, 6-8 time-points with bal-

anced sampling may be appropriate. For small molecules (≤1,000 Da), 1, 2, 4, and 8 hr. sampling

point recommended.66 The animal experiment was carried out on SD rat. In study protocol, 10 μL

optimized formulation was instilled into rat eyes. At 0.5, 1, 2, 4, and 8 h after the instillations (N=3

rats/time point), blood samples were withdrawn in microfuge tubes containing K2EDTA (20

μL/mL of blood, 200 mM) as anticoagulant from tail vein and the eyes were removed followed by

euthanasia at respective time points. The plasma samples were harvested from blood by centrifu-

gation of samples at 7500 RPM for 10 min at 4°C and stored below -60°C until bioanalysis.

Ocular tissue samples were weighed and homogenized using a pre-chilled micro homogenizer.

Protein extraction reagent was added with a ratio of tissue to reagent of 1:5 (1g of tissue/5 ml of

reagent). The lysed samples were centrifuged for 10,000 rpm at 40C for 10 min and supernatant

was transferred to a chilled EPP tube.67 The resultant samples were analyzed by developed and

validated HPLC method.

3.14 Stability Studies

3.14.1 Accelerated Stability Study by Centrifugation Stress Test

Stress stability study of the microemulsion formulation was carried out by subjecting it to centrif-

ugation. The formulation was centrifuged at 9,000 rpm for 20 min by Centrifuge (Make Remi) and

examined for phase separation.68

3.14.2 Stability Study as per ICH guidelines

The stability of the microemulsion and microemulsion based gel were assessed under different

storage conditions as per International Conference of Harmonization (ICH ) guidelines, at room



temperature (250C ± 20C / 60% RH ± 5% RH), at refrigeration condition (2-80C/45% RH ± 5%

RH), at 400C ± 20C / 75% RH ± 5%RH.69

The samples of microemulsion were evaluated at 0, 1, 2, 3 and 6 months for pH, viscosity, globule

size, zeta potential. Further, the samples of microemulsion based gel were evaluated at 0, 1, 2, and

3 months for pH, viscosity and drug content. All the stability parameters were determined in tripli-

cate.

3.15 Data Analysis

Pseudoternary phase diagrams were plotted using Prosim software. The data obtained were

analyzed statistically using t-test and ANOVA. The data obtained for the optimization of pro-

cess and formulation variables were statistically analyzed by analysis of variance (ANOVA)

with in-built software design of Design Expert 7.0.0 software (Stat-Ease, Inc., USA). It was

used to determine the significance and the magnitude of the effects of different variables and

their interactions. Probability values less than 0.0500 were considered as statistically signifi-

cant. Pharmacokinetic data fitted to PK Solver 2.0 software for determination various parame-

ters.

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44.

CHAPTER 4 Results and Discussion


CHAPTER 4

Results and Discussion



4.1 Identification of Drug

4.1.1 Identification of Drug by Physical Attributes

Before preceding the formulation development, identification of drug and ensuring its purity is

prerequisite. The identification test of ebastine based on physical examination like appearance,

solubility and melting point are given in following Table 4.1

TABLE 4.1 Identification test for ebastine with the standard/ inference

Sr.

No. Properties Specification

1 Observation Inference

1. Appearance White or almost

white powder White powder Complies

2. State Crystalline Crystalline Complies

3. Melting Point 80-820C 84-87

0C Complies

4.

Solubility

Practically in-

soluble in water,

freely soluble in methanol

Practically in-

soluble in water,

freely soluble in methanol

Complies

Table 4.1 shows that all the observations in term of melting point, solubility and physical ex-

amination like color, state complies with their reported specifications. This indicates the purity

of procured drug sample.

4.1.2 Identification of Drug by FTIR

Fourier Transform Infrared spectroscopy (FTIR) study of drug sample was performed to de-

termine various functional groups into it. FTIR spectra was obtained and compared with refer-

ence spectra available in literature.2 The obtained and reference spectra of ebastine are shown

in Fig 4.1

The major IR peaks obtained and reported for ebastine molecule (as shown in Fig 4.2) is sum-

marized in Table 4.2



FIGURE 4.1 (a) Observed IR spectra of ebastine

FIGURE 4.1 (b) Reference IR spectra of ebastine



FIGURE 4.2 Structure of ebastine

TABLE 4.2 Interpretation of FTIR spectra of ebastine

Observed (cm-1

) Reported (cm-1

) Inferences3

3084, 3052 3000-3100 CH-Aromatic

2955,2943

2854 -2926 CH-Aliphatic

1678 1670-1820 C=O (Ketone)

1604, 1452 1495 -1562 C=C (Aromatic)

1359 1350-1000 C-N

1070 1300-1000 C-O

Interpretation of FTIR spectra of ebastine suggest that major peaks observed in procured as

well as reference spectra. The observations provide confirmation of purity of sample.



4.1.3 Identification of Drug by DSC

The DSC thermogram exhibit the idea about crystalline and amorphous state of sample. It also

interpret any type of incompatibility or interaction, if persist amongst ingredients of formula-

tion.4 The thermogram of pure ebastine shown in Fig 4.3 exhibited a sharp endothermic peak at

88.690C equivalent to its reference melting point 80-82

0C, representing crystalline state of drug

sample.

FIGURE 4.3 DSC Thermogram of ebastine

4.2 Analytical Method

To determine drug content and drug release, simple, rapid, accurate and reliable analytical

method is needed during preformulation, formulation and optimization stage. The analytical

methods were also required for the in-vivo kinetic study for estimation of concentration of drug

in ocular tissue and plasma.



4.2.1 UV Spectrophotometric Estimation of Ebastine

Development and validation of simple, accurate and rapid UV spectrophotometric analytical

methods have been reported for the estimation of ebastine in formulation and stability sam-

ples.5,

6

Hence, calibration curves were obtained using methanol and methanolic phosphate buffered

saline (PBS), pH 7.4, 30% v/v as solvents for determining the solubility of drug in microemul-

sion components and in vitro release studies.

4.2.1.1 Calibration Curve in Methanol as Solvent

Calibration curve was plotted in methanol in the range of 2-10µg/ml of ebastine at 252 nm us-

ing UV spectrophotometer (UV-1800, Shimadzu) for determining the solubility of drug and

drug content. The calibration curve data is shown in Table 4.3 and the overlay spectra of ebas-

tine at different concentrations and the calibration curve is depicted in Fig. 4.4 (a) and (b) re-

spectively.

TABLE 4.3 Calibration curve data for ebastine in methanol as solvent

Sr. No. Conc. (mcg/ml) Absorbance

(Mean ± SD)

1 2 0.044 ± 0.001

2 4 0.172 ± 0.007

3 6 0.290 ± 0.001

4 8 0.397 ± 0.078

5 10 0.512 ± 0.004



FIGURE 4.4 (a) Overlay spectra of ebastine at different concentrations

FIGURE 4.4 (b) Calibration curve for ebastine in methanol as solvent

The calibration plot was found to be linear in concentration range of 2-10 µg/ml of ebastine

with correlation coefficient value (R2) of 0.9990



4.2.1.2 Calibration Curve in Methanolic Phosphate Buffer (PBS, pH 7.4, 30% v/v) as Sol-

vent

Calibration curve was plotted in methanolic phosphate buffer in the range of 10-30µg/ml of

ebastine at 254 nm using UV spectrophotometer (UV-1800, Shimadzu) for determining drug

release profile. The calibration curve is shown in Table 4.4 and the overlay spectra of ebastine

at different concentrations and the calibration curve is depicted in Fig. 4.5 (a) and (b) respec-

tively.

TABLE 4.4 Calibration curve data for ebastine in methanolic PBS (pH 7.4, 30% v/v) as

solvent

Sr No Conc. (mcg/ml) Absorbance

(Mean ± SD)

1 10 0.148 ± 0.009

2 15 0.253 ± 0.005

3 20 0.361 ± 0.011

4 25 0.478 ± 0.001

5 30 0.605 ± 0.002

FIGURE 4.5 (a) Overlay spectra of ebastine at different concentrations



FIGURE 4.5 (b) Calibration curve for ebastine in in methanolic PBS (pH 7.4, 30% v/v) as

solvent

The calibration plot was found to be linear in concentration range of 10-30 µg/ml of ebastine

with correlation coefficient value (R2) of 0.9984. The pH was selected based on the ocular site

pH 6.5-7.6. Ebastine has limited solubility in buffer but is soluble in methanol, hence meth-

anolic PBS pH 7.4 used to maintain the perfect sink conditions.7,

8

4.2.2 HPLC Method Development and Validation

4.2.2.1. RP- HPLC Method development for Pure Ebastine

Standard solution of ebastine (100 ppm) was scanned in the range of 200-400 nm and from that

the detection wavelength (λmax) was selected at 244 nm. Different mobile phases were tried to

obtained good peak shape with sufficient theoretical plates and minimum asymmetry. Mobile

phase was selected that give shorter run time, retention time, more than 2000 theoretical plates

and less than 1.5 tailing factor and reproducible results. Summary of all mobile phase trials are

reported in Table 4.5.



Table 4.5 Trials for selection of mobile phase

Trial Mobile phase

1 Methanol: Water ( 80:20 %v/v)

2 Methanol: Water: Acetic acid (70:20:10 %v/v)

4 Acetonitrile: Water (50:50 %v/v)

5 Acetonitrile: Water (80:20 %v/v) pH adjusted 2.5 with glacial acetic acid

6 Acetonitrile: water: methanol (70:20:10 %v/v)

7 Acetonitrile: water: methanol (70:20:10 %v/v) pH adjusted 2.5 with glacial ace-

tic acid

8 Methanol: acetonitrile: ammonium acetate buffer (80:10:10)

9 Methanol: acetonitrile: ammonium acetate buffer (80:10:10), pH adjusted 5.5

with glacial acetic acid

Various trials were taken by using different columns and mobile phase, by changing the pH of

the mobile phase to select the appropriate chromatographic conditions giving good resolving

peak characteristics. The optimized chromatographic conditions were,

Elution: Isocratic

Column/ Stationary phase: Waters X-Terra Shield, Phenomenex C18 (250 mm x 4.5

mm, 5 µm particle size)

Mobile phase: Methanol: Acetonitrile: Ammonium acetate buffer (80:10:10), pH ad-

justed 5.5 with glacial acetic acid

Flow rate: 1.2 ml/min

Detection wavelength: 244nm

Injection volume: 20µL

Run time: 10 min.

a. System Suitability Test

System suitability parameters like theoretical plates, tailing factor and retention time were

studied by injecting 6 replicates of standard stock concentration (10 µg/ml of Ebastine) and



%RSD for retention time was calculated. Chromatogram of system suitability is shown in

Fig.4.6 and Table 4.6 mentioned the data of system suitability.

FIGURE 4.6 Chromatogram of system suitability

TABLE 4.6 Data of system suitability for ebastine (10 µg/ml)

Sr. No. Retention time(min) Theoretical plates Tailing factor

1 7.363 2009 1.687

2 7.347 2035 1.678

3 7.320 2056 1.647

4 7.320 2056 1.686

5 7.330 2061 1.686

6 7.350 2073 1.634

Mean 7.33833333 Theoretical plates

are more than 2000

Tailing factor is less than

2 S.D. 0.01767107

%RSD 0.24080493

Tailing factor for ebastine was found to be less than 2 which depicts that shape of ebastine

peak was symmetrical. Theoretical plates were found to more than 2000 and % RSD of reten-

tion time is less than 2 which show the column efficiency. The developed analytical method

validated as per ICH guidelines.9

b. Linearity and Range

For underlying study, the calibration curve of ebastine was obtained in range of 2-10µg/ml.

The good linear relationship in the range of the calibration curve constructed was showed by

the linear regression data. The value of correlation coefficient (r2) was found to be 0.9993 indi-

cating that method is linear with straight line equation of y = 34.561x+19.191. The overlay



chromatogram obtained for linearity study is shown in Fig. 4.7 (a) and the calibration plot with

regression equation and correlation coefficient was determined as shown in Fig. 4.7 (b).

FIGURE 4.7 (a) Chromatogram for linearity study

FIGURE 4.7 (b) Calibration plot for linearity study

c. Repeatability

Standard stock solution (10 µg/ml of ebastine) was injected six times, area of each peak was

measured and %RSD was calculated. The data of repeatability is shown in Table 4.7



TABLE 4.7 Data of repeatability

Sr. No. Concentration (µg/ml) Area

1 10 127.918

2 10 127.907

3 10 128.299

4 10 125.489

5 10 126.905

6 10 124.863

Mean 126.8968

S.D. 1.424628

%RSD 1.122667

As depicted in above table, %RSD found to be 1.122667%, which was less than 2 indicating

that the developed method is precise.

d. Precision

Interday Precision

Three concentrations of working standard solutions of ebastine 4, 6, 8µg/ml were analyzed at

three consecutive days and %RSD was calculated. The data of interday precision is shown in

Table 4.8.

TABLE 4.8 Data of interday precision

Concentration

(µg/ml)

Area

MEAN

S.D.

%RSD Day 1 Day 2 Day 3

4 90.151 91.156 88.357 89.888 1.4179129 1.57742179

6 123.839 125.996 124.878 124.9043 1.07874109 0.86365385

8 156.107 154.978 157.134 156.073 1.07840206 0.69096004



Intraday Precision

Three concentrations of working standard solutions of ebastine 4, 6, 8 µg/ml were analyzed at

three times on the same day and %RSD was calculated. The data of intraday precision is

shown in Table 4.9.

TABLE 4.9 Data of intraday precision

Concen-

tration

(µg/ml)

Area

MEAN

S.D.

%RSD Time 1 Time 2 Time 3

4 90.151 92.153 91.153 91.152 1.41562778 1.55304083

6 123.839 126.012 124.674 124.9255 1.53654304 1.22996749

8 156.107 154.078 155.386 155.0925 1.43471966 0.92507353

The % RSD values of inter and intraday precision lies within the acceptance limit concluding

that method is precise.

e. Accuracy

Accuracy was determined by taking known drug sample at 50, 100 and 150% level of the spec-

ification was spiked to 4µg/ml. The amount of ebastine was calculated at each level and % re-

coveries were computed. The data of accuracy reported is shown in Table 4.10.

TABLE 4.10 Data of accuracy

Level

Amount of

sample

(µg/ml)

Amt of std.

spiked

(µg/ml)

Total

amount

(µg/ml)

Total Mean recov-

ered amount

(µg/ml)

%recovery

50% 4 2 6 5.9505 99.1750

100% 4 4 8 8.0734 100.9186

150% 4 6 10 10.0526 100.5260

Accuracy is the degree of agreement between the observed value and true value. The accuracy

of developed method was assessed by performing recovery study. The mean % recovery at

three different concentrations was found to be 99.1750%, 100.9186% and 100.5260% at 50%,



100% and 150% respectively. The results of recovery study fall within the acceptance limit

indicating that developed method is accurate.

f. Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD is the minimum concentration of an analyte that can be detected by developed method

and LOQ is the lowest concentration of an analyte that can be quantified by method with accu-

racy and precision. LOD and LOQ were found by injecting lower concentrations like

0.1μg/mL, 0.01μg/mL, 0.001μg/mL, 0.0001 μg/mL. At 0.001μg/mL, the peak is detectable

which is considered as LOD. At 0.003μg/mL (3:1, Signal: Noise), the drug was quantifiable

with suitable accuracy and precision, hence 0.003 μg /mL was considered as LOQ.

g. Robustness

Robustness of the developed method was assessed to determine the capacity of method to re-

main less/ unaffected by small, but purposely changes brought about in various method param-

eters. Robustness is criteria of the reliability of method during its routine use. In this, three

concentrations of working standard solutions of ebastine 4, 6, 8µg/ml was analyzed at three

different wavelengths (243nm, 244nm, 245nm) and %RSD was calculated. The data of robust-

ness is shown in Table 4.11

.

TABLE 4.11 Data of robustness at different wave length

Concentration

(µg/ml)

Area MEAN S.D. %RSD

243nm 244nm 245nm

4 90.151 89.156 92.001 90.436 1.44375 1.5964

6 125.839 123.996 124.02 124.618 1.0572 0.8483

8

156.107

153.978

155.134

155.073

1.06581

0.6873

The %RSD of different wavelength was found to be 0.6873-1.5964%. It was found to be less

than 2 indicating that developed method is robust.



4.2.2.2 Bioanalytical Method Development in Ocular Tissue

The goal of this work was to develop and validate a simple, rapid and sensitive assay method

for quantitative determination of drug by extraction of ocular tissues. Chromatographic condi-

tions, especially the composition and acidic nature of the mobile phase, were optimized to

achieve best resolution. It also increased the signal to noise ratio and to minimize run times. In

the present study, phenylephrine hydrochloride was used as an internal standard (ISTD) as

comparatively it was very well resolved from ebastine with sharp symmetrical peak shape

among all the trials. For extraction of ebastine from ocular tissue, acetonitrile was used to pre-

cipitate protein. The following chromatographic conditions were selected for the determination

of ebastine in ocular tissue,

Elution: Isocratic

Column/ Stationary phase: Waters X-Terra Shield, Phenomenex C18 (250 mm x 4.5

mm, 5 µm particle size)

Mobile phase: Methanol: Acetonitrile: Ammonium acetate buffer (80:10:10), pH ad-

justed 5.5 with glacial acetic acid

Flow rate: 1.2 ml/min

Detection wavelength: 244nm

Injection volume: 20µL

Run time: 10 min.

Optimized chromatographic parameter was found to be similar as that of pure drug while dif-

ferent trials for development of analytical method for estimation ebastine in tissue matrix with

different internal standards were taken; few representive trials are shown in Table 4.12 and Fig.

4.8 (a), (b) and (c).



TABLE 4.12 Different experimental trials for RP-HPLC method development

Trials Mobile Phase ISTD Remarks

Trial-1

Methanol:ACN:Ammonium

acetate buffer (80:10:10)

Pheniramine

maleate alone

Peak shape of Pheniramine

maleate was not proper.

Trial-2


acetate buffer (80:10:10)

Phenylephrine

HCL+ Drug

All system suitability param-

eters are in limit.

Trial-3


acetate (80:10:10)

Phenylephrine

HCL + Drug

+Tissue

All three Peaks of drug,

ISTD and tissue were well

separated.

Trial 1:

RP-HPLC Trial of Pheniramine maleate (ISTD) and Ebastine

FIGURE 4.8 (a) Chromatogram of Pheniramine maleate (ISTD)

Trial 2:

RP-HPLC Trial of Phenylephrine Hydrochloride (ISTD) and Ebastine

FIGURE 4.8 (b) Chromatogram of Phenylephrine Hydrochloride (ISTD) and Ebastine



Trial 3:

RP-HPLC Trial of Phenylephrine Hydrochloride (ISTD), ocular tissue and Ebastine

FIGURE 4.8 (c) Chromatogram of phenylephrine hydrochloride (ISTD) and ebastine in

tissue matrix

a. System Suitability Test for Drug with Internal Standard

System suitability is the test for checking of a system to ensure system performance prior to or

during the analysis of unknown sample. For system suitability test, six chromatogram were tak-

en and observed the parameters like theoretical plate count, tailing factors, resolution and re-

producibility in retention time (RT). The %RSD was calculated. The chromatogram is recorded

in Fig.4.9 and the data of system suitability is shown in Table 4.13.

FIGURE 4.9 Chromatogram of system suitability of ebastine and ISTD



TABLE 4.13 System suitability parameters of chromatogram for ebastine & ISTD

Inj

no.

RT –

EBA

RT-

ISTD

RESOLUTION

-EBA

Tailing Factor Theoretical Plate

EBA ISTD EBA ISTD

1 7.34 4.06 6.56 1.945 1.421 2532 2501

2 7.347 4.063 6.53 1.972 1.482 2488 2503

3 7.287 4.033 6.696 1.944 1.473 2595 2609

4 7.283 4.037 6.682 1.957 1.411 2645 2567

5 7.353 4.07 6.642 1.972 1.446 2591 2550

6 7.397 4.107 6.543 1.959 1.431 2522 2536

AVG 7.3345 4.06166

Greater

than 2

Less

than 2

Less

than 2

Greater

than

2000

Greater

than 2000

SD 0.0432284 0.02668

RSD 0.5893852 0.65689

Less than 2

Less than 2


The linearity of an analytical procedure is its ability (within a given range) to obtain test results

which are directly proportional to the concentration (amount) of analyte in the sample. Calibra-

tion curve was constructed by plotting the peak area ratios (analyte/internal standard) v/s con-

centration. The overlay chromatogram obtained for linearity study is shown in Fig. 4.10 (a) and

the calibration plot with regression equation and correlation coefficient was determined as

shown in Fig. 4.10 (b). The calibration plot revealed linearity in the concentration range of 3-

400 ng /ml with the correlation coefficient of 0.991. Table 4.14(a) and 4.14(b) indicate the

sample preparation and area ratios obtained for linearity study respectively.

TABLE 4.14 (a) Sample preparation for linearity study of drug with ISTD

Standard

Working Stock

conc.(µg/ml)

Volume of stock

solution (in µl)

Volume

make up to

Final

Conc.

(ng/ml)

Std 1 100 40 10ml 400

Std 2 100 36 10ml 360

Std 3 100 30 10ml 300



Std 4 100 20 10ml 200

Std 5 100 6 10ml 60

Std 6 10 20 10ml 20

Std 7 10 6 10ml 6

Std 8 10 3 10ml 3

TABLE 4.14(b) Concentration, area and area ratio for linearity study of drug with ISTD

Sr No.

Concentration

of EBA (ng/ml)

Area of

EBA

Area of ISTD

Area of EBA

Area of ISTD

1 3 14.314 41 0.349122

2 6 28.271 42.152 0.670692

3 20 32.597 41.014 0.794777

4 60 43.012 41.079 1.047056

5 200 85.668 41.903 2.044436

6 300 115.161 41.86 2.751099

7 360 127.319 41.372 3.07742

8 400 150.165 41.53 3.61582

FIGURE 4.10 (a) Chromatogram for linearity study of drug with ISTD



FIGURE 4.10 (b) Calibration plot for linearity study of drug with ISTD

C. Extraction Trials in Ocular Tissue10,

11

Trial 1:

A 500 µl ocular sample was aliquoted into 5ml eppendorf tube .After that 50µl ISTD was

added, spiked required volume of standard solution of required concentration. All samples

were treated with 1 ml of acetone, vortexed for 2 min and centrifuged for 10min at 5000

RPM at 40C. The supernant was transferred to test tube and injected in to HPLC system.

Trial 2:

A 500 µl ocular sample was aliquoted into 5ml eppendorf tube. After that 50 µl ISTD was

added, spiked required volume of standard solution of required concentration. All samples

were treated with 1 ml of ethyl acetate, vortexed for 2 min and centrifuged for 10 min at

5000 RPM at 40C. The supernant was transferred to test tube and injected in to HPLC sys-

tem.

Trial 3:

A 500 µl ocular sample sample was mixed with 50 µl of ISTD working solution and spiked

required concentration standard solution of drugs. To this, 1 ml of acetonitrile was added.



After vortex mixing for 2 min and centrifuging at 5000 RPM for 10min at 40C. The super-

nant was transferred to test tube and injected to HPLC system. The observed data for all tri-

als with justification are given below in Table 4.15. The representive chromatogram of drug

in ocular tissue with ISTD is depicted in Fig.4.11.

TABLE 4.15: Data obtained for extraction trails in ocular tissue

Extraction method

Trials

Protein pre-

cipitation

Result

Protein Precipita-

tion method

Trial 1 Acetone Interference of solvent

was observed

Trial 2 Ethyl Acetate Low recovery of ana-

lytes

Trial 3 (Op-

timized

Trial)

Acetonitrile

Good recovery of ana-

lytes and ISTD.

FIGURE 4.11: Chromatogram of drug with ISTD in ocular tissue

Extraction method with acetonitrile as precipitating agent was selected as it gives maximum

recovery as compare to other two solvents.

d. System Suitability Test for Drug with Internal Standard in Ocular Tissue

System suitability is the checking of a system to ensure system performance prior to or during

the analysis of unknowns. For system suitability test the mixture was prepared containing solu-

tion of 4 µg /ml, 8 µg /ml, 12 µg /ml, 4 µg /ml of ebastine and ISTD respectively. The solu-

tions were extracted from ocular tissue by protein precipitation method. After that the six



chromatogram were taken and the parameters were observed such as theoretical plate count,

tailing factors, resolution and reproducibility in retention time (RT).The % RSD was calculat-

ed. The data for system suitability of drug with ISTD in ocular tissue is shown in Table 4.16.

Fig. 4.12 showed that all the peaks were well resolved.

FIGURE 4.12: Chromatogram of system suitability of drug with ISTD in ocular tissue

TABLE 4.16: System suitability parameters of drug with ISTD in ocular tissue

e. Sensitivity

Sensitivity was carried out by injecting repeatedly Lower Limit of Quantification (LLOQ)

which is the lowest concentration of the standard curve and that can be measured with accepta-

ble accuracy and precision. The 6 replicates of extracted LLOQ samples (mixture of 0.003 and

0.025µg/ml of ebastine and ISTD respectively) were injected repeatedly at the same chromato-

Inj

no.

RT-

EBA

RT-

ISTD RESOLUTION

-EBA


EBA EBA ISTD

1 7.777 4.257 6.456 1.670 1.075 2812 2206

2 7.810 4.290 6.293 1.595 1.17 2574 2636

3 7.827 4.280 6.472 1.784 1.115 2727 2455

4 7.810 4.267 6.272 1.619 1.157 2597 2361

5 7.737 4.237 6.453 1.720 1.204 2687 2571

6 7.780 4.253 7.566 1.162 1.326 2790 2424

RSD :

0.415831

<2

RSD :

0.4498

9039< 2

RESOLUTION

>2

Tailing

Factor

<2

Tailing

Factor

<2

Theoret-

ical

Plate >2000

Theoret-

ical

Plate >2000



graphic condition and measurements of retention time, peak area, mean and % CV were calcu-

lated. The data of sensitivity is shown in Table 4.17.

TABLE 4.17 Sensitivity data for LLOQ samples

ISTD EBA

RT Area RT Area

1 4.316 15.59 7.075 9.276

2 4.329 13.15 7.391 8.26

3 4.239 17.491 7.14 9.226

4 4.239 11.831 7.377 8.98

5 4.329 10.999 7.375 6.487

6 4.330 12.457 7.47 7.524

%CV 0.9518 18.1794 1.7631 13.3887

It is observed from result table that %CV of RT less than 2 and area for LLOQ sample was

observed within the acceptance range.

f. Linearity and Range

The calibration curve was containing at least 8 non-zero samples, zero sample (matrix

processed with Internal standard) and blank matrix sample. Eight non-zero samples of

0.0625-8 µg/ml and 0.09375-12 µg/ml ebastine and ISTD respectively and each pro-

cessed with 4 µg/ml of ISTD and inject. The blank and zero samples were injected and

analyzed to confirm the absence of direct interference. The non-zero samples analyzed

and plotted. The calibration curve was prepared by area ratio v/s concentration. Table

4.18 indicates area ratios obtained for linearity study in ocular tissue. The overlay chro-

matogram obtained for linearity study is shown in Fig.4.13 (a) and the calibration plot

with regression equation and correlation coefficient was determined as shown in Fig. 4.13

(b).



TABLE 4 .18 Concentration, area and area ratio for linearity study of drug with ISTD

in ocular tissue

Sr No.

Concentration

of EBA (ng/ml)

Area of EBA

Area of ISTD

Area of EBA

Area of ISTD

1 3 7.314 33.633 0.217465

2 6 24.271 33.881

0.71636

3 20 28.597 32.878 0.869791

4 60 39.012 32.691 1.193356

5 200 81.668 33.643 2.427489

6 300 114.161 34.763 3.28398

7 360 123.319 32.981 3.739092

8 400 146.165 33.803 4.324024

FIGURE 4.13 (a) Overlay chromatogram for linearity study of drug with ISTD in ocular

tissue



FIGURE 4.13 (b) Calibration plot for linearity study of drug with ISTD in ocular tissue

g. Accuracy

Intraday accuracy was determined by analyzing five replicates of three concentration levels

(LQC, MQC, HQC) at three different times of the same day. Inter-day accuracy was deter-

mined by analyzing five replicates of three different concentrations, (LQC, MQC and HQC)

on three consecutive days. The data of accuracy is reported in Table 4.19.

TABLE 4.19 Data of accuracy for ebastine

QC

Samples

Conc.

(µg/ml)

Intraday %Accu-

racy

Interday %Accu-

racy

LQC 0.009 88.19% 89.12%

MQC 0.2 91.08% 91.72%

HQC 0.3 91.28% 90.49%

Intraday accuracy & Interday accuracy for ebastine was observed from 88.19%% to 91.28%

and 89.12% to 91.72% respectively which are within the acceptance range.

h. Precision

The precision of an analytical procedure expresses the closeness of agreement (degree of scat-



ter) between a series of measurements obtained from multiple sampling of the same homoge-

neous sample under the prescribed conditions. Intra-day precision was determined by analyz-

ing five replicates of three different concentrations, (LQC, MQC and HQC) on a single day.

Inter-day precision was determined by analyzing five replicates of three different concentra-

tions, (LQC, MQC and HQC) on three consecutive days. The data of precision is reported in

Table 4.20.

TABLE 4.20 Data of precision for ebastine

QC

Samples

Conc.

(µg/ml)

% CV

(Intra-day precision)

% CV

(Inter-day precision)

LQC 0.009 7.8925 7.6895

MQC 0.2 5.7594 8.1787

HQC 0.3 6.7259 6.6719

The precision determined at each concentration level should not exceed 15% of the coefficient

of variation (%CV) except for the LLOQ, where it should not exceed 20% of the %CV. The

%CV of Intraday precision & Interday precision for HQC, MQC and LQC was observed to be

within acceptance criteria. The data thus obtained demonstrates that method is accurate and

precise for the quantification of ebastine from ocular tissue.

i. Recovery

The extraction recovery was performed to evaluate extraction procedure used to extract ebas-

tine from ocular tissue by developed bioanalytical method. The % recovery determined at

LQC, MQC and HQC are given in Table 4.21 (a), (b) and (c) respectively.



TABLE 4.21 (a) Data of recovery at LQC

Sr. No

LQC

Area Ratio of Extracted

Area ratio

post extracted % Recovery

1 0.6923 0.7791 88.86%

2 0.6715 0.7801 86.08%

3 0.6801 0.7681 88.55%

4 0.6690 0.7698 86.91%

5 0.6714 0.7618 88.13%

6 0.6800 0.7519 90.44%

Mean 0.677389483 0.768466667 88.16%

SD 0.008686909 0.010669708 0.01531038

%CV 1.282409778 1.388441212 1.73665105

TABLE 4.21 (b) Data of recovery at MQC

Sr. No

MQC

Area

Ratio of Extracted

Area ratio

post extracted

%Recovery

1 2.2272 2.4012 92.75%

2 2.1487 2.3123 92.92%

3 2.1479 2.4012 89.45%

4 2.3442 2.5012 93.72%

5 2.0472 2.2012 93.00%

6 2.2478 2.4005 93.64%

Mean 2.19383333 2.3696 92.58%

SD 0.10225859 0.1018944 0.01584

%CV 4.66118304 4.30006737 1.71097



TABLE 4.21 (c) Data of recovery at HQC

Sr. No

HQC

Area

Ratio of Extracted

Area ratio

post extracted

%Recovery

1 2.9272 3.2688 89.55%

2 2.8787 3.2674 88.10%

3 2.8981 3.2582 88.95%

4 2.8429 3.2782 86.72%

5 2.9478 3.2682 90.20%

6 2.9471 3.2699 90.13%

Mean 2.906966667 3.26845 88.94%

SD 0.041640349 0.00638 0.013404

%CV 1.432432972 0.195196 1.5071

The overall mean recovery of ebastine was found to be 88.16%, 92.58%, 88.94% at LQC,

MQC and HQC level respectively. The % recovery was observed within range of 15% for

MQC, HQC and within range of 20% for LQC which is within acceptance criteria, indicat-

ing that said extraction procedure used was suitable for measurement of ebastine from tis-

sue matrix.

j. Stability Study

Short term stability, freeze thaw stability, long term stability and stock solution stability

studies were performed as per USFDA Guidelines. The data obtained for different stability

studies are shown in Table 4.22 (a-e)



TABLE 4.22(a) Data of short term stability

QC Samples LQC HQC

Sr. No. Area ratio Area ratio

Fresh After 6 hr. Fresh After 6 hr.

1 0.7211 0.7211 3.2840 3.2713

2 0.7212 0.7205 3.2784 3.2111

3 0.7245 0.7239 3.2814 3.2287

4 0.7262 0.7252 3.2849 3.2647

5 0.7242 0.7231 3.2365 3.2145

Mean 0.72344 0.72276 3.27303602 3.23806

SD 0.002225534 0.00195141 0.02057994 0.02821397

%CV 0.307632097 0.269994154 0.62877223 0.8713232

%Stability 99.91% 98.93%

TABLE 4.22 (b) Data of freeze thaw stability

QC Samples LQC HQC


Fresh After 3 cycles Fresh After 3 cycles

1 0.7211 0.7094 3.2840 3.1771

2 0.7212 0.7145 3.2784 3.2711

3 0.7245 0.7116 3.2814 3.1719

4 0.7262 0.7187 3.2849 3.2814

5 0.7242 0.7131 3.2365 3.2241

Mean 0.72344 0.71346 3.27303602 3.22512

SD 0.002225534 0.003486115 0.02057994 0.05104147

%CV 0.307632097 0.48862099 0.62877223 1.58262246

%Stability 98.62% 98.54%



TABLE 4.22 (c) Data of long term stability

QC Samples LQC HQC


Fresh After 7 days Fresh After 7 days

1 0.7211 0.7012 3.2840 3.0547

2 0.7212 0.6991 3.2784 3.0158

3 0.7245 0.6956 3.2814 3.0147

4 0.7262 0.7084 3.2849 3.099483

5 0.7242 0.7019 3.2365 3.2145

Mean 0.72344 0.70124 3.27303602 3.0798366

SD 0.002225534 0.004692867 0.02057994 0.08290887

%CV 0.307632097 0.669224078 0.62877223 2.69198929

%Stability 96.93% 94.10%

TABLE 4.22 (d) Stock solution stability at room temperature after 6 hours

QC Samples LQC HQC


Fresh After 6 hr. at

RT

Fresh After 6 hr. at

RT

1 0.7211 0.7059 3.2840 3.1543

2 0.7212 0.7029 3.2784 3.1147

3 0.7245 0.6989 3.2814 3.0258

4 0.7262 0.6859 3.2849 3.1268

5 0.7242 0.7126 3.2365 3.1549

Mean 0.72344 0.70124 3.27303602 3.1153

SD 0.002225534 0.009928645 0.02057994 0.05299061

%CV 0.307632097 1.415869806 0.62877223 1.70097944

%Stability 96.93% 95.18%



TABLE 4.22 (e) Data of stock solution stability at refrigerated condition (2-8˚C) for 7

days

QC Sam-

ples LQC HQC

Sr. No.

Area ratio Area ratio

Fresh After 7 days at 2-8˚C Fresh After 7 days at

2-8˚C

1 0.7211 0.6933 3.2840 3.1478

2 0.7212 0.6881 3.2784 3.1258

3 0.7245 0.7021 3.2814 3.1879

4 0.7262 0.6981 3.2849 3.1873

5 0.7242 0.6925 3.2365 3.142

Mean 0.72344 0.69482 3.27303602 3.15816

SD 0.002225534 0.005399259 0.02057994 0.0280591

%CV 0.307632097 0.777073085 0.62877223 0.88846348

%Stability 96.04% 96.49%

The data obtained from different stability studies showed that % stability of the mean calcu-

lated concentration of fresh and stability quality control samples found to be ≤ 15% of nomi-

nal value which is within acceptance criteria.

4.2.2.3 Bioanalytical Study of Plasma Sample12

Plasma samples are also subjected to analytical evaluation for determination of drug concentra-

tion using developed HPLC method. As the undertaken drug delivery is categorized under top-

ical, it is necessary to determine of drug concentration in plasma hypothesizing that negligible

or very less drug reaches systemic drug delivery, proving topical utility of undertaken formula-

tion

All chromatographic parameters are same as the method of estimation of drug in tissue except

flow rate. It was from 1.2 ml/min to 0.8 ml/min because at this flow rate, plasma peak was well

resolved from ISTD peak.



FIGURE 4.14 Chromatogram of drug with ISTD in plasma

a. System Suitability Test for the Drug with Internal Standard in Plasma

System suitability is the checking of a system to ensure system performance prior to or during

the analysis of unknowns. For system suitability test the mixture was prepared containing of

ebastine and ISTD. The solutions were extracted from plasma by protein precipitation meth-

od. After that the six chromatogram were taken and the parameters were observed such as

theoretical plate count, tailing factors, resolution and reproducibility in retention time (RT).

The % RSD was calculated. The data for system suitability of drug with ISTD in plasma is

shown in Table 4.23. Fig. 4.15 showed that all the peaks were well resolved.

FIGURE 4.15 Chromatogram of system suitability of drug with ISTD in plasma



TABLE 4.23 Data of system suitability parameters of ebastine with ISTD in plasma

Inj.

no.

RT -

EBA

RT -

ISTD

RESOLUTION

-EBA


EBA ISTD EBA ISTD

1 7.383 4.030 5.456 1.770 1.171 2793 2206

2 7.343 4.090 5.293 1.695 1.173 2674 2536

3 7.297 4.093 5.472 1.711 1.126 2797 2451

4 7.343 4.087 5.272 1.627 1.174 2591 233

5 7.313 4.083 5.453 1.711 1.217 2787 2557

6 7.313 4.051 5.566 1.693 1.296 2799 2410

RSD :

0.4221<2

RSD :

0.6323<2

RESOLUTION

>2

Tailing

Factor

<2

Tailing

Factor

<2

Theoretical

Plate

>2000

Theoretical

Plate >2000


The calibration curve was containing at least 8 non-zero samples, zero sample (plasma matrix

processed with Internal standard) and blank plasma matrix sample. Eight non-zero samples of

0.012-1.560 µg/ml ebastine and 4 µg/ml of ISTD were inject. The blank and zero samples were

injected and analyzed to confirm the absence of direct interference. The non-zero samples ana-

lyzed and plotted. The calibration curve was prepared by area ratio v/s concentration. Table

4.24 indicates area ratios obtained for linearity study in plasma. The overlay chromatogram

obtained for linearity study is shown in Fig.4.16 (a) and the calibration plot with regression

equation and correlation coefficient was determined as shown in Fig. 4.16 (b).



TABLE 4.24 Concentration, area and area ratio for linearity study drug with ISTD in

plasma

Sr

No.

Concentration of

EBA(µg/ml)

Area of EBA

Area of ISTD

Area of EBA

Area of ISTD

1 0.012 41 8.314 0.20278

2 0.024 42.152 24.271 0.575797

3 0.078 41.014 32.597 0.794777

4 0.234 41.079 48.012 1.168772

5 0.780 41.903 90.668 2.163759

6 1.170 41.86 114.161 2.72721

7 1.404 41.372 127.319 3.07742

8 1.560 41.53 150.165 3.61582

FIGURE 4.16(a) Overlay chromatogram for linearity study of drug with ISTD in plasma



FIGURE 4.16(b) Calibration plot for linearity study of drug with ISTD in plasma

The developed method validated for different validation parameters like accuracy, precision,

sensitivity, recovery and stability.

4.3 Screening of Components of Microemulsion

The most primary criteria for selection of the components were that all the components are

pharmaceutically acceptable for ocular administration and fall under GRAS (generally recog-

nized as safe) category. The major components for microemulsion formulation include the

drug, oil, surfactants and cosurfactants.13

After performing preliminary identification test on

drug, various components were screened for further proposed formulation. The rationale be-

hind selection based on maximum solubility of drug, compatibility and safety status.

4.3.1 Selection of Oil

The selection of the oil was primarily depends on the solubility of the drug in oil in order to

increase maximum drug loading potential. The solubility of drug was determined in eight dif-

ferent oils- Campul MCM EP, Oleic acid, Isopropyl palmitate, Arachis oil, Linseed oil, Light

liquid paraffin, Labrafac and ethyl oleate. The result obtained are summarized in Table 4.25



and Fig 4.17.

TABLE 4.25 Selection of oil based on solubility of drug in oil

Sr No Oil Solubility (mg/ml)

Mean ± SD

1 Capmul MCM EP 28.5 ± 0.2

2 Oleic acid 15 ± 0.1

3 Isopropyl palmitate 10 ± 0.11

4 Arachis oil 08 ± 0.2

5 Linseed oil 11.5 ± 0.3

6 Light liq. Paraffin 14.3 ± 0.12

7 Labrafac 11.6 ± 0.2

8 Ethyl Oleate 12.4 ± 0.92

FIGURE 4.17 Solubility of ebastine in different oils

Amongst various oils tested, ebastine showed maximum solubility in Campul MCM EP (28.5

± 0.2 mg/ ml). While light liquid paraffin and ethyl oleate showed next highest solubilizing

capacity of 14.3 ± 0.12 mg/ml, 12.4 ± 0.92mg/ml respectively. Campul MCM EP is reported

to be used for ophthalmic microemulsion formulation.14,15

High molecular weight oil exhibit

difficulty in formation of microemulsion as they contain long chain fatty acids which shows

difficulty in penetration of the interfacial film formed by surfactant and cosurfactant which



assist in optimal curvature formation. Campul MCM EP is a mono-diglyceride of medium

chain fatty acids (mainly caprylic and capric) and categorized as small molecular weight tri-

glycerides.

4.3.2 Selection of Surfactant

The most critical problem related to the microemulsion formulation is the toxicity of the sur-

factants that may cause ocular irritation. Therefore, it is very important to select suitable sur-

factant as well as use the minimum concentration of it in the formulation. Non-ionic surfac-

tants are comparatively less toxic and have minimum irritation potential than their ionic coun-

terpart. They are also less affected by pH and changes in ionic strength. Another important

parameter need to consider during selection of surfactant is the HLB value.16,

17

Once oil has

been selected, next goal was to identify the surfactant that has highest capacity for solubiliza-

tion of drug. In this study, various surfactants were tried for maximum solubility potential of

drug- Labrasol, Tween 80, tween 20, Span 80, Cremophore EL, and Lauroglycol FCC. The

results are summarized in Table 4.26 and Fig. 4.18

TABLE 4.26 Selection of surfactant based on solubility of drug in surfactant

Sr No Surfactant Solubility (mg/ml)

Mean ± SD

1 Labrasol 23.1 ± 0.3

2 Tween 80 19.8 ± 0.1

3 Tween 20 12.3 ± 0.4

4 Span 80 9.2 ± 0.21

5 Cremophor EL 14.7 ± 0.29

6 Lauroglycol FCC 15.5 ± 0.58



FIGURE 4.18 Solubility of ebastine in different surfactants

Ebastine shown maximum solubility in Labrasol (23.1 ± 0.3mg/ml) followed by Tween 80

(19.8 ± 0.1mg/ ml) Therefore, blend of Labrasol and tween 80 was selected as the surfactant

for microemulsion formulation. Among the all classes of surfactants, nonionic surfactants are

considered more versatile because of their improved solubilization characteristics, nonirritancy

and ability to prolong precorneal retention with enhanced permeability.

4.3.3 Selection of Co-surfactant

Beside selection of surfactant, one more important consideration in the formulation of Micro-

emulsion is the flexibility of the interface to promote the Microemulsion formation. With the

view, surfactant(s) are combinely used with a cosurfactant. The penetration of cosurfactant into

the interfacial film provide more fluid interface thereby allowing the free movement of hydro-

phobic tails of the surfactants at the interface.18,19

The solubility of drug was determined in six

different cosurfactants-propylene glycol, isopropyl alcohol, ethanol, glycerol, butanol and PEG

400. The results are summarized in Table 4.27 and Fig. 4.19.



TABLE 4.27 Selection of co-surfactant based on solubility of drug in co-surfactant

Sr. No. Co-Surfactant Solubility (mg/ml)

Mean ± SD

1 Propylene glycol 18.2 ± 0.1

2 Isopropyl alcohol 14.7 ± 0.2

3 Ethanol 12.9 ± 0.11

4 Glycerol 11.9 ± 0.25

5 Butanol 13.7 ± 0.36

6 PEG 400 11.1 ± 0.72

FIGURE 4.19 Solubility of ebastine in different co-surfactants

Amongst co surfactant tested, maximum solubility of drug obtained in propylene glycol (18.2 ±

0.1mg/ml) followed by alcohols. Amongst various S mix blends, propylene glycol, isopropyl

alcohol and ethanol forms transparent system with 98.21%T, 99.01%T, and 99.17% T respec-

tively compared to other tested cosurfactants with selected surfactant. Due to ocular compatibil-

ity and volatility issue, selection of any alcohol as microemulsion component was prohibited.

Therefore, blend propylene glycol and glycerol was selected as the co-surfactant for microemul-

sion formulation. Moreover, glycerol will help in maintaining osmolarity of formulation.20



4.3.4 Drug Excipient Compatibility Study

IR spectra of pure drug ebastine, optimized formulation and physical mixture of ebastine and

gelling polymer, methyl cellulose, sodium hyaluronate are shown in Fig. 4.20.

a. Drug + Oil (Campul MCM EP) b. Drug+ surfactant ( Labrasol)

c. Drug+ surfactant (Tween 80) d. Drug+ co surfactant (propylene glycol)

e. Drug+ co surfactant (Glycerol) f. Optimized Microemulsion formulation

FIGURE 4.20 IR spectra for drug excipient compatibility



It was observed from Fig.4.20, that all characteristic peaks of the ebastine were found in the IR

spectra of screened excipient and drug mixture as well as optimized formulation indicating the

compatibility/no interaction of the drug with the screened excipients.

4.3.5 Differential Scanning Calorimetry Study

The Differential scanning calorimetry (DSC) thermograms are taken to characterize the physical

state of drug in the polymer matrix, temperature dependent conformational changes and to as-

sess incompatibility, if any. In the present work, thermal behavior of drug and physical mixture

of drug and polymer determined by DSC. The thermogram of pure ebastine (Fig 4.3 as shown

previous) exhibited a sharp endothermic peak at 88.690C equivalent to its reference melting

point 80-820C, representing crystalline state of drug sample. The thermogram of physical mix-

ture of polymer (sodium hyaluronate and carboxyl methyl cellulose) and drug (ebastine) showed

the peaks at 252.30 °C and 273.64 °C. Appearance of no new peak and absence of any potential

shift in peak of drug in physical mixture shown in Fig.4.21 indicates compatibility of ebastine with

polymers and was confirmed FTIR as well as DSC study

FIGURE 4.21 DSC Thermogram of physical mixture (drug + polymers



4.4 Construction of Pseudo- ternary Phase Diagrams

Pseudoternary phase diagrams for Individual Smix system and blend Smix system were con-

structed as per method described in methodology section. Individual Smix system ratios (1:1,

2:1 and 3:1) employed Campul MCM EP as oil, Labrasol as surfactants and propylene glycol as

co-surfactant. While, blend Smix system ratios 2(1:1):1 employed mixture of surfactants

(Labrasol +Tween 80) and 2(1:1): 1(1:1) employed mixture of surfactants (Labrasol +Tween

80) and mixture of co-surfactants (Propylene glycol + Glycerol).

Fig. 4.22 (a) and (b) illustrated pseudo ternary phase diagram for individual Smix system and

blend Smix system respectively. The preliminary batches for construction of pseudo ternary di-

agram with percentage composition of each component of microemulsion at various oil to Smix

ratio, individual and blend both the systems are shown in Table 4.28(a) and (b).

TABLE 4.28 (a) Trial compositions of preliminary pseudo ternary batches of Oil: Smix In-

dividual system

Oil: Smix; 1:1

Batch

No.

Oil : Smix

Ratio(w/w)

(1:1)

Water uptake

after 24 hr.

equilibrium

Oil

(% w/w)

Smix

(% w/w)

Water

(%w/w)

1 1:9 0.55 7.84 70.59 21.57

2 2:8 0.52 15.87 63.49 20.63

3 3:7 0.51 23.90 55.78 20.32

4 4: 6 0.5 32.00 48.00 20.00

5 5 : 5 0.48 40.32 40.32 19.35

6 6: 4 0.45 48.98 32.65 18.37

7 7 :3 0.43 57.61 24.69 17.70

8 8:2 0.42 66.12 16.53 17.36

9 9:1 0.39 75.31 8.37 16.32



Oil: Smix; 2:1

Batch

No.

Oil : Smix

Ratio(w/w)

(2:1)

Water uptake

after 24 hr.

equilibrium

Oil

(% w/w)

Smix

(% w/w)

Water

(%w/w)

10 1:9 0.57 7.78 70.04 22.18

11 2:8 0.53 15.81 63.24 20.95

12 3:7 0.5 24.00 56.00 20.00

13 4: 6 0.51 31.87 47.81 20.32

14 5 : 5 0.49 40.16 40.16 19.68

15 6: 4 0.45 48.98 32.65 18.37

16 7 :3 0.45 57.14 24.49 18.37

17 8:2 0.43 65.84 16.46 17.70

18 9:1 0.41 74.69 8.30 17.01

Oil: Smix; 3:1

Batch

No.

Oil : Smix

Ratio(w/w)

(3:1)

Water uptake

after 24 hr.

equilibrium

Oil

(% w/w)

Smix

(% w/w)

Water

(%w/w)

19 1:9 0.6 7.69 69.23 23.08

20 2:8 0.58 15.50 62.02 22.48

21 3:7 0.55 23.53 54.90 21.57

22 4: 6 0.54 31.50 47.24 21.26

23 5 : 5 0.52 39.68 39.68 20.63

24 6: 4 0.5 48.00 32.00 20.00

25 7 :3 0.48 56.45 24.19 19.35

26 8:2 0.47 64.78 16.19 19.03

27 9:1 0.45 73.47 8.16 18.37



TABLE 4.28 (b) Trial compositions of preliminary pseudo ternary batches of Oil: Smix

Blend system

{Oil: Smix; 2(1:1):1}

Batch

No.

Oil :Smix

Ratio(w/w)

{2(1:1):1}

Water uptake

after 24 hr.

equilibrium

Oil

(% w/w)

Smix

(% w/w)

Water

(%w/w)

28 1:9 Indefinite - - -

29 2:8 2.6 8.70 34.78 56.52

30 3:7 2.1 14.63 34.15 51.22

31 4: 6 1.9 20.51 30.77 48.72

32 5 : 5 1.8 26.32 26.32 47.37

33 6: 4 1.6 33.33 22.22 44.44

34 7 :3 1.4 41.18 17.65 41.18

35 8:2 1.2 50.00 12.50 37.50

36 9:1 0.9 62.07 6.90 31.03

{Oil: Smix; 2(1:1):1(1:1)}

Batch

No.

Oil :Smix

Ratio (w/w)

{2(1:1):1(1:1)}

Water uptake

after 24 hr.

equilibrium

Oil

(% w/w)

Smix

(% w/w)

Water

(%w/w)

37 1:9 indefinite - - -

38 2:8 4.8 5.88 23.53 70.59

39 3:7 3.9 10.17 23.73 66.10

40 4: 6 3.7 14.04 21.05 64.91

41 5 : 5 3.2 19.23 19.23 61.54

42 6: 4 2.9 24.49 16.33 59.18

43 7 :3 2.2 33.33 14.29 52.38

44 8:2 1.7 43.24 10.81 45.95

45 9:1 1.4 52.94 5.88 41.18



FIGURE 4.22 (a) Pseudo Ternary Diagram of Oil: Smix individual system (1:1), (2:1),

(3:1), composed of oil (Campul MCM EP), surfactant (Labrasol) and co-surfactant (pro-

pylene glycol)

FIGURE 4.22 (b) Pseudo Ternary Diagram of Oil: Smix Blend system 2(1:1):1, composed

of oil (Campul MCM EP), surfactants (Labrasol +Tween 8) and co-surfactant (propylene

glycol); 2(1:1): 1(1:1), composed of oil (Campul MCM EP), surfactants (Labrasol +Tween

8) and co-surfactants (propylene glycol+ Glycerol)



Among the various combinations of individual Smix system (i.e. 1:1, 2:1, 3:1) explored, the

maximal region for microemulsion was observed at the ratio of 2:1 among all individual Smix

system. Hence, selected individual Smix system ratio was further studied by blend Smix system

ratios, 2(1:1):1, using mixtures of surfactants and 2(1:1): 1(1:1), using mixtures of surfactants

and co-surfactants.

A higher microemulsion region was observed in blend Smix system as compared to individual

Smix system. The probable reasons are a reduction of the interfacial tension by surfactant and

increased the fluidity of the interface by cosurfactant. According to the pseudo ternary phase

diagram, the blend Smix ratio 2(1:1): 1(1:1) produced broader microemulsion region was se-

lected for further formulation optimization.

4.5 Optimization of Microemulsion by D-Optimal Mixture Design

In the present study, D-optimal mixture experimental design was applied. Formulation variables

were Campul MCM EP (X1), Smix (X2), and water (X3) while, Globule size (nm) (Y1), Viscosi-

ty (cp) (Y2) and Transmittance (%) (Y3) were assessed as response variables. The data obtained

from globule size in nm (response Y1), viscosity in cp (response Y2), and transmittance in %

(response Y3) was analyzed using Design Expert® Software. The compositions of design ma-

trix batches with measured responses are given Table 4.29.

TABLE 4.29 Compositions of design matrix batches with responses

Batches

(ME)

A:Oil

(gm)

B:Smix

(gm)

C:Water

(gm)

Globule

Size (nm)

Viscosity

(cps)

Transmittance

(%)

ME1 4 3.139 6.861 311 10.9 97.2

ME2 2 3.428 8.572 213.1 9.98 98.4

ME3 3.861 4.742 5.397 213.5 12.74 99

ME4 2.001 2 9.999 395.1 9.5 98

ME5 3.115 6 4.885 143.2 13.63 99

ME6 2.234 6 5.766 176.1 12.91 99.7



ME7 2.001 2 9.999 395.1 9.12 98

ME8 3.998 6 4.002 135.3 14.14 99.1

ME9 3.064 3.102 7.834 236.1 10.53 98.6

ME10 2.007 4.29 7.703 182.1 11.67 99.2

ME11 2.946 4.127 6.927 210.8 12.23 99

ME12 3.505 2 8.495 354.7 9.47 98.4

The polynomial equations constitute the coefficients for intercept, main first-order effects, in-

teraction term. The coefficients value exhibits the effect of these formulation variables on the

response. A positive sign of coefficient indicates a synergistic effect while negative term indi-

cates an antagonistic effect on the response.21

The data summarized in Table 4.30.

TABLE 4.30 Coefficients of cubic equation for each independent variable

Coefficient Globule size (nm) Transmittance (%) Viscosity (cps)

A(Oil) +12332.37 -17.42 +319.48

B(S mix) -183.07 +96.87 +0.16

C(Water) +395.14 +98.00 +9.28

AB -21162.35 +222.24 -528.76

AC -20873.16 +188.96 -549.36

BC +267.36 +9.88 +29.37

ABC +19659.51 -211.96 +512.18

AB (A-B) -11469.5 +122.26 -338.12

AC (A-C) -9475.61 +65.83 -274.17

BC(B-C) +991.69 +12.02 +23.55

After generating the polynomial equations through MLRA (Multiple linear regression analysis)



relating the dependent and independent variables, mixture components were optimized for the

responses. The values of all the responses were fitted to models viz. linear, quadratic, special

cubic and cubic model where the best fit model was found to be cubic model for all the re-

sponses as compared to other models as shown in Table 4.31.

TABLE 4.31 Summary of regression analysis for all responses

Model Std. Dev. R-Squared Adjusted R-

Squared

Predicted

R-Squared Remark

Response 1 Globule size(nm)

Linear 41.60808297 0.837278282 0.8011179 0.715878822

Quadratic 32.84677275 0.932394153 0.876055946 0.63422757

Special Cubic 14.55714352 0.988934528 0.975655962 0.936588798

Cubic 1.573384878 0.999948293 0.999715613 0.881835238 Suggested

Response 2 Viscosity (cps)

Linear 0.356569823 0.965192595 0.957457616 0.941721624

Quadratic 0.364390049 0.975766049 0.955571089 0.933770144

Special Cubic 0.392523532 0.976566273 0.948445801 0.904700682

Cubic 0.321333363 0.993718236 0.9654503 0.954526667 Suggested

Response 3 Transmittance (%)

Linear 0.438370154 0.65729547 0.581138908 0.327795929

Quadratic 0.476286834 0.730298239 0.505546771 -0.52049235

Special Cubic 0.459194362 0.791090361 0.540398794 -0.90850877

Cubic 0.024307431 0.999765845 0.998712147 0.464988912 Suggested

R

2 values were reported resemble to unity indicating the high predictive ability of Response

Surface Methodology (RSM) of underlying study. Further, the higher values (>4) of ―Adequate

Precision‖ indicate adequate signal for suitable model selection.22

Fig. 4.23 (a-i, ii), 4.23 (b-i, ii) and 4.23 (c-i, ii) shows contour, 3D response curve for depend-

ent variables viz. globule size (nm), viscosity (cps) and transmittance (%) respectively. It can

be observed from the response variables plots of Globule size that as the concentration of oil



increases, globule size also increases while the concentration of Smix increase then globule

size decreases. It can be observed from the response variables plots of viscosity that as the

concentration of Smix increases and decrease in amount of water, viscosity increases. It can be

observed from the response variables plots of transmittance that as the concentration of oil in-

creases % transmittance decreases and Smix increases % transmittance increases. Moreover,

linear correlation was found analogous for actual response and predicated response {Figure

4.23 (a-iii), 4.23 (b-iii), 4.23 (c-iii)}. The reliability of these response surfaces was also con-

firmed by the corresponding residual plot between the experimental run and the internally stu-

dentized residuals for all response variables, as shown in Figure {Figure 4.23 (a-iv), 4.23 (b-

iv), 4.23 (c-iv) } The vertical distribution of the internally studentized residuals was in line

from top to bottom under the completely randomized run. These findings revealed that all

points fall within a confidence interval of 95 %. The ANOVA analysis for globule size, viscos-

ity, % transmittance shown in Table 4.32 (a), (b) and (c).

TABLE 4.32(a) Data of ANOVA table for globule size (Y1)

ANOVA for Response Surface Cubic Model

Source Sum of

Squares

df Mean

Square

F

Value

p-value

Prob > F

Model 95748.05 9 10638.67 4297.52 0.0002 significant

Linear Mix-

ture 80171.91 2 40085.95 16192.81 < 0.0001

AB 486.77 1 486.77 196.63 0.005

AC 468.72 1 468.72 189.34 0.0052

BC 43.71 1 43.71 17.66 0.0522

ABC 459.21 1 459.21 185.5 0.0053

AB(A-B) 399.41 1 399.41 161.34 0.0061

AC(A-C) 399.22 1 399.22 161.26 0.0061

BC(B-C) 974.32 1 974.32 393.58 0.0025

Residual 4.95 2 2.48

Lack of Fit 4.95 1 4.95



FIGURE 4.23 (a) Response variable globule size (Y1)

i. Contour plot ii. Surface response curve

iii. Predicted Vs. Actual response

iv. Residual Vs. run

iv

iii

ii

i

C: Water

B: Smix

357.728

Globule Size

395.1

135.3

X1 = A: Oil

X2 = B: Smix

X3 = C: Water

A: Oil 8



TABLE 4.32(b) Data of ANOVA table for viscosity (Y2)


Source

Sum of

Squares

df

Mean

Square

F

Value

p-value

Prob > F


Linear Mix-

ture 31.73 2 15.87 153.65 0.0065

AB 0.3 1 0.3 2.94 0.2284

AC 0.32 1 0.32 3.14 0.2182

BC 0.53 1 0.53 5.11 0.1522

ABC 0.31 1 0.31 3.02 0.2245

AB(A-B) 0.35 1 0.35 3.36 0.2082

AC(A-C) 0.33 1 0.33 3.24 0.2138

BC(B-C) 0.55 1 0.55 5.32 0.1474

Residual 0.21 2 0.1

Lack of Fit 0.13 1 0.13 1.86 0.4028 not signif-

icant



FIGURE 4.23 (b) Response variable viscosity (Y2)




iv

iii

Viscosity:

14.14

9.12

Viscosity

ii

X1 = A: Oil

X2 = B: Smix

9.12

8.000

B: Smix i

9.12

X1 = A: Oil X2 = B: Smix

X3 = C: Water

A: Oil



TABLE 4.32(c) Data of ANOVA table for % transmittance (Y3)


Source Sum of

Squares

df Mean

Square

F

Value

p-value

Prob > F


Linear Mix-

ture 3.32 2 1.66 2807.1 0.0004

AB 0.054 1 0.054 90.86 0.0108

AC 0.038 1 0.038 65.02 0.015

BC 0.06 1 0.06 100.95 0.0098

ABC 0.053 1 0.053 90.34 0.0109

AB(A-B) 0.045 1 0.045 76.81 0.0128

AC(A-C) 0.019 1 0.019 32.61 0.0293

BC(B-C) 0.14 1 0.14 242.14 0.0041

Residual 1.18E-03 2 5.91E-04

Lack of Fit 1.18E-03 1 1.18E-03



FIGURE 4.23 (c) Response variable % transmittance (Y3)




iv

iii

Color points by value of

Transmittance:

99.7

97.2

Transmittance

ii

C: Water

8.000

B: Smix i

97.4572

99.181 98.6064

99.7

97.2

X1 = A: Oil

X2 = B: Smix

X3 = C: Water

A: Oil



4.5.1 Experimental Validation of Design Space

Experimental validation of DoE trials for formulation variables was undertaken by formulation

and characterization of microemulsion formulation at the check point batch suggested by the

software. Figure 4.24 shows the overlay plot displaying the design space and optimized param-

eters as check point suggested by DoE software to obtain the desired responses. The observed

values were comparable with the predicted values establishing the reliability of the optimiza-

tion procedure as shown in Table 4.33. Calculated percentage prediction error was found to be

less than 5 percent, confirming the validity of D- optimal mixture design for microemulsion

formulation optimization.

Figure 4.24 Overlay plot

TABLE 4.33 Checkpoint analysis of optimized formulation

Parameters Predicted value Experimental value % Error

Globule Size (nm) 143.33 142 ± 0.16 0.92

Viscosity(cps) 13.51 13.19± 0.121 2.36

Transmittance (%) 99.09 99.79± 0.134 0.70

Error (%) = (predicted value – experimental value)/ predicted value × 100.

Data expressed were of mean ±SEM (n=3)



4.6 Formulation and Optimization of Microemulsion Based Gel

The topical application of optimized microemulsion is restricted due to low viscosity resulted

in less residential time of formulation. To overcome this, as well as for maintaining the ease of

formulation application, gelling agents were incorporated into formulation. The ocular delivery

improved by adding mucoadhesive polymer in formulation. The weight ratio of CMC (1%) and

SH (1.5%) was found satisfactory based on proper gel formation. The former polymer used in

commercial ocular formulations, as it has desirable mucoadhesive and a high retention time on

the ocular surface and the latter one exhibit excellent viscoelastic, lubricating and water reten-

tion properties. The literature reveled that, this combination benefited with high viscosity under

low friction conditions (between blinking) which stabilizes the tear film and low viscosity un-

der high friction conditions (during the blinking) which reduces discomfort in animal as well as

humans23

.

A specified amount of drug consisting of the chosen oil and Smix was magnetically stirred un-

til the drug completely dissolved; microemulsion was prepared by adding aqueous phase. 1%

CMC and 1.5% SH polymers dispersion was formed by suspending in water. The polymer dis-

persion kept for overnight to form viscous gel matrix. Prepared microemulsion and polymer

dispersion was mixed in 1:1 v/w ratio 24

. Smooth viscous, transparent gel was formed. The trial

batches conducted for selection of gelling/ mucoadhesive polymers are shown in Table 4.34.

While, Table 4.35 depicted different trials taken for preparation of microemulsion based gel

formulation. All the characteristic peak of pure ebastine (CH-Aromatic, CH-Aliphatic, C=O (Ketone),

C=C (Aromatic), C-N, C-O) observed in IR spectra of physical mixtures. Hence, no significant in-

compatibility was seen between drug and the screened excipients as observed with IR spectra

of pure drug ebastine, physical mixtures of ebastine with polymers and that of with preserva-

tive. (shown in Fig. 4.25).



TABLE 4.34 Different trial batches for selection of gelling/ mucoadhesive polymers

Sr.

No. Trials Observations

1 100 mg chitosan + 10 ml SWI Magnetic stirring Insoluble

2 100 mg chitosan + 10 ml SWI (heat) Magnetic Stirring No proper gel

3 100 mg chitosan+10ml warm SWI (400C) Magnetic

Stirring

Hydrated, lump

formation

4 100 mg chitosan + Buffer pH 7.4 Magnetic stirring Insoluble

5 100 mg chitosan + 0.1 M Acetic acid Magnetic stirring Gel formation,

pH shifted to

acidic (pH 5.9)

6 100 mg CMC ++ 10 ml SWI Magnetic stirring Clear Gel for-

mation

7 100 mg Sodium Hyaluronate ++ 10 ml SWI Magnetic

stirring

Clear Gel For-

mation

TABLE 4.35. Different trials batches for microemulsion based gel

Sr Microemulsion

(g) CMC (% w/v) SH (% w/v) Observation

1 1 Part 1 0.5 Less viscous

2 1 Part 1 1 Moderate viscous

3 1 Part 1 1.5 Highly viscous , poura-

ble

4 1 Part 1 2 Highly viscous , sticky,

Non pourable



(b) Physical mixture of drug and polymers (c) Physical mixture of drug and preservative

FIGURE 4.25 IR Spectra for drug excipient compatibility

(a) IR spectra of drug (ebastine) (refer Fig. 4.20) (b) IR spectra of physical mixture of

drug and polymers (ebastine+ Carboxyl methyl cellulose + Sodium Hyaluronate) (c) IR

spectra of physical mixture of drug and preservative (Sodium perborate)

4.7 Evaluation of Optimized Microemulsion Formulation

4.7.1 pH

The pH of the optimized formulation was determined to be 6.9 ± 0.12, which can be easily

buffered by tear fluid (pH 6.5-7.6) which indicated non irritancy of formulation; hence it is ad-

equate to instilled into the eye without reflex tear and rapid tear blinking. Moreover, the eye

can tolerate pH of 6.5 -8.0 without any discomfort.

4.7.2 Droplet size, Zeta Potential and Viscosity Measurement

The globule size that human eyes can tolerate is about 10 micrometer

25, indicating suitability

of developed formulation for ocular use. The Polydispersity Index (PDI) was found to be well

below 1.0 which confirms monodispersed (narrow size distribution) nature of formulation, it

also indicate that the optimized microemulsion remains stable upon dilution. Zeta potential is

crucial physicochemical parameter that correlated with stability of dispersed system. Zeta po-

tential determines aggregation potential of the globules. The droplet size of optimized micro-

emulsion formulation was determined by Malvern Zetasizer Nanoseries Nano –ZS and found

to be 142± 0.16 nm as shown in the Figure 4.26. Zeta potential of optimized microemulsion

formulations was found to be -22.6 ± 0.39 mV as shown in the Figure 4.27. Viscosity is im-



portant parameter need to be assessed since highly viscous formulation exhibit interference

with normal functioning like blinking, vision, ease of application etc., while very less viscous

formulation will show lack of residential time at the site of instillation. The Viscosity of opti-

mized formulation was found to be 13.19 ± 0.121cps at room temperature. Owing to less vis-

cosity, the residential capacity of formulation at physiological site (eye) can be increased by

adding gelling agent.

FIGURE 4.26 Globule size measurement of optimized formulation



FIGURE 4.27 Zeta potential measurement of optimized formulation

4.7.3 Measurement of Refractive Index

Refractive index measurement used to detect possible vision impairment or discomfort to the

applicant after application of ocular formulation.26

Refractive index of tear fluid is 1.340 to

1.360. It is recommended that ocular formulation should have refractive index values not high-

er than 1.476. The optimized formulation had refractive index values 1.369 ±0.04 which is re-

semble to the recommended values.

4.7.4 Measurement of Osmolarity

Isotonicity of ophthalmic preparations is of prime consideration.

27 The osmolarity of tear film

in human after prolonged eye closure is 288-293 mOsm/L and as eye is open, it progressively

rises up to 302-318 mOsm/L.25

An Osmolarity of optimized formulation was found to be 291 ±

0.301mOsm/L indicating appropriateness for ocular application. The glycerol used in underly-

ing formulation performed dual role of imparting osmolarity to formulation and act as a

cosurfactant also.



4.7.5 Measurement of Surface Tension

Ophthalmic formulation should have the surface tension range at the surface to air interface of

34.3-70.9mN/m. The surface tension of the optimized microemulsion formulation was found to

be 34.75 ±0.13 mN/m. Low microemulsion surface tension ensures good spreading effect on

the conjunctive, cornea and mixing with precorneal film components, thereby improving con-

tact between the drug and the conjunctival tissue.28

4.7.6 Drug Content

Microemulsion of ebastine with blend of surfactant and cosurfactant were prepared by Phase

Titration Method (Water titration) method. The percentage of drug content of optimized for-

mulations was found to be 97.09 ± 0.12%

4.7.7 Transmission Electron Microscopy

The surface morphology of the droplets of optimized formulation measured using Transmis-

sion electron microscope (TEM). The pictorial image taken using transmission electron micro-

scope with CCD camera (TEM Philips Tecnai 20, Holland). The Figure 4.28 showed spherical

shape and uniform droplet size of optimized microemulsion. As the loaded ebastine micro-

emulsion globules are nanometric and morphologically spherical, they are not expected to

cause ocular irritation.



FIGURE 4.28 Transmission Electron Microscopy (TEM) of optimized formulation

4.7.8 Measurement of % Transmittance

Microemulsion loaded with ebastine diluted with distilled water for 10 times. The % transmit-

tance was found 99.79 ± 0.134 with reference to purified water as blank at 650 nm using UV

spectrophotometer. (UV-1800, Shimadzu)

4.8 Evaluation of Microemulsion Based Gel

4.8.1 pH

The pH of the 1% w/v aqueous solution of the prepared microemulsion based gels was meas-

ured by digital pH meter. The pH value was found to be is 6.8 ± 0.44.



4.8.2 Rheology Study

The Rheograms of microemulsion gel and microemulsion based gel diluted with tear fluid in a

ratio of 40:7 were determined at different shear stress using Plate and cone viscometer. Proper

spread of the gels on the ocular surface will ensure increased absorption of the drug after ocu-

lar administration. The pseudo plastic character of precorneal tear film should be disturbed

less/or not disturbed by the administration of ophthalmic products. The ocular shear rate is

about 0.03 s-1 during interblinking periods and 4250 – 2850 s-1 during blinking. The viscoe-

lastic fluid having high viscosity under low shear rates providing a higher retention time for

therapeutic treatment and low viscosity under high shear rates providing comfort, called as

pseudo plastic fluid, is often preferred for ophthalmic application. The Rheogram of micro-

emulsion gel and microemulsion gel diluted with tear fluid (To mimic physiological condition,

formulations were mixed with artificial tear fluid (ATF) in a ratio of 40:7) exhibited pseudo-

plastic behavior, i.e., decrease in the viscosity with increase in angular velocity exhibiting its

suitability for ophthalmic use. The Rheogram of microemulsion gel and microemulsion gel

shear rate and shear stress are depicted in Fig. 4.29 (a) and (b).



FIGURE 4.29 (a) Rheogram of microemulsion based gel



FIGURE 4.29 (b) Rheogram of microemulsion based gel diluted with tear fluid



4.8.3 Mucoadhesive Strength

Modified two-pan balance method was used to determine mucoadhesive strength

29. 15.7 ml of

water was required to be added to detach the cellophane membrane from the gel. Hence, the

mucoadhesive force was calculated as 15,401.7 dynes/cm2 using Equation

F = w x g

=15.7 x 981 dynes/cm2

= 15,401.7 dynes/cm2

Where F is the mucoadhesion force (dynes / cm2),

w is the minimum weight required to break the bond (grams), g

is the acceleration due to gravity (cm/s2).

4.8.4 Spreadability

The gel spreadability was measured by placing 0.5 g gel between two cellophane membranes

and placing 100gm weight on it for one min. The spreadability of the gel was found to be 2.8

cm/gm gel. Uniform and effective spreading of the gel on ocular surface will ensure increased ab-

sorption of drug after ocular application.

4.8.5 Drug Content Determination

The optimized microemulsion formulation converted into final microemulsion based gel for-

mulation. The ebastine content in the optimized microemulsion based gel formulation was

found to be 98.22 ± 0.40 %

4.8.6 In vitro Drug Release Study

In vitro release profiles of drug loaded microemulsion and microemulsion based gel were de-

termined by dialysis bag method shown in Fig. 4.30. It is difficult to mimic diffusion cell in

vitro method with the real situation in vivo because cellulose membrane cannot exhibit the bar-

riers of ocular multilayered epithelium as well as the constant volume of diffusion cell will not



be able to eliminate the drug released by tear fluid turnover and nasolacrimal leakage. This

phenomenon affects the concentration gradient and the diffusion of the drug through the epi-

thelium. Therefore, possibility exists that formulations would have a different release in vivo.

FIGURE 4.30 In vitro release profiles of microemulsion and microemulsion based gel;

data expressed were of percent Mean ± SEM

Drug release profile depicted in figure shown maximum % ebastine released from micro-

emulsion was found 89.19 ± 2.45% compared to microemulsion based gel 71.34 ± 2.34%

within 8 hr. However, microemulsion gel was able to sustain the release of the remaining

ebastine for up to 24 hr. It is found that drug release from microemulsion is comparative-

ly more than microemulsion based gel. This might be possible matrix effect on release of

ebastine due to incorporation of microemulsion in CMC and HA gel, a micro gel layer

forms around the droplets that can hinder drug diffusion from the oil phase, so the rate

and the amount of the released drug may decrease, while the release rate of the drug from



microemulsion depends on the rate of diffusion of the drug from oil droplets. The possi-

bility of the drug partition between the oil and the water phases in the presence of the sur-

factant positioned at the oil–water interface prior to release. Formulation provided the

highest in vitro drug release with the ability of providing a sustained release over 24 hr.,

thus reducing frequency of application and improving patient compliance.

4.8.7 Kinetics of Drug Release Study

The mechanism of drug release from ebastine loaded microemulsion and microemulsion based

gel was determined from in vitro data. The obtained data was fitted to various kinetic models

(Zero order, First order, Higuchi model, Korsmeyer peppas model) and the best fit model was

obtained by regression analysis. Fig. 4.31 (a), (b), (c), (d) and 4.32 (a), (b) , (c), (d) depicts

graphs for various kinetics model fitting for microemulsion and microemulsion based gel for-

mulation respectively.



a. Zero order release kinetics b. First order release kinetics

c. Higuchi Model of release kinetics d. Korsemeyer -Pepppa’s Model of release

FIGURE 4.31 Models for drug release kinetic (Microemulsion)



a. Zero order release kinetics b. First order release kinetics

c. Higuchi Model of release kinetics d. Korsemeyer -Pepppa’s Model of release

FIGURE 4.32 Models for drug release kinetic (Microemulsion based gel)

The in vitro release studies data was fitted into various mathematical models to determine the

best fit model as shown in Table 4.36

TABLE 4.36 Regression coefficients for release kinetics

Release Kinetic of Microemulsion

Zero order re-

lease

First order

release

Higuchi

model

Korsmeyer peppas Release mecha-

nism

R 2 R 2 R 2 N R 2 Anomalous

0.8662 0.958 0.971 0.76 0.978 transport

Release Kinetic of Microemulsion based gel

Zero order re-

lease

First order

release

Higuchi

model

Korsmeyer peppas Release mecha-

nism

R 2 R 2 R 2 N R 2 Anomalous

0.943 0.988 0.970 0.43 0.959 transport



The R2 value is considered as the tool for representing the best fitting of kinetic model. Ac-

cording to data obtained from in vitro release study of ebastine from microemulsion shows Hi-

guchi Patten and the release mechanism expected to be anomalous diffusion. The release of

dug from microemulsion based gel best fits in the first order release kinetics indicating concen-

tration dependent diffusion controlled release pattern. The results were found in accordance

with investigations by other researchers.30, 31

, 32

4.9 Sterilization and Sterility Testing

Sterility testing is crucial requisite for clinical use of an ophthalmic drug delivery. The various

methods used are moist heat sterilization, gamma radiation and sterile filtration. Of all meth-

ods, sterile filtration method predominantly used for chemically or thermally sensitive materi-

al. Sterilization of prepared microemulsion formulation was carried out by membrane filtra-

tion using 0.22μm membrane filter. Sterility testing of optimized microemulsion formulation

was performed by direct inoculation method. The result of sterility testing is shown in Table

4.37 (a), (b) and Fig. 4.33.

TABLE 4.37 (a) Results of sterility testing in FTGM (Fungi)

No. of

days 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Sample -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

Negative control

-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

Positive control

-ve -ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve

FTGM: Fluid Thioglycolate Medium

TABLE 4.37 (b) Results of sterility testing in SCDM (Bacteria)

No. of

days

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Sample -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

Negative control

-ve

-ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve -ve

Positive

control

-ve -ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve

SCDM: Soyabean Casein Digest Medium



FIGURE 4.33 Day 1: Sterility testing set up of optimized microemulsion formulation

using soybean- casein digest medium and fluid thioglycolate medium

From the result Table 4.37(a) and (b), we can conclude that optimized microemulsion

formulation was sterile. No sign of growth in the negative control confirmed the sterility of

fluid thioglycolate medium and soyabean-casein digest medium. Presence of turbidity in the

positive control confirmed that medium is capable of inducing a growth. No growth in sample

confirmed the sterility of formulation. To avoid contamination in final gel product, necessary

protocol carried out in aseptic cabinet.



4.10 In vitro / In vivo studies

4.10.1 Ocular Irritation Study by Hen’s Egg Chorioallantoic Membrane (HET-CAM)

Test

HET- CAM (Hen‗s chorioallantoic membrane) experiment used for testing the potential of op-

timized formulation for eye irritation as per literature.33

The effects of optimized formulation

as well as positive and negative controls on the chorioallantoic membrane were noted before

and after the treatment as shown in Fig 4.34. There was a remarkable difference between the

optimized formulation and positive control by observing the changes in the chorioallantoic

membrane. The positive control (0.1 N NaOH) induced major damage to the CAM. The 0.1 N

NaOH causes hemorrhage followed by the lysis of blood vessels, whereas optimized formula-

tion and negative control (0.9% NaCl) does not show severe changes in the chorioallantoic

membrane after the application. The severity of ocular irritation of the formulations was com-

pared with that of positive and negative controls. The images showed that there was no consid-

erable change in the blood vessel morphology of isolated CAM and formulation did not cause

any damage or irritation to the eye upon application.



Before Treatment 5 min After Treatment

a. Optimized formulation a. Optimized formulation

b. Positive Control (0.1N NaOH) b. Positive Control (0.1N NaOH)

c. Negative Control (0.9% NaCl) c. Negative Control (0.9% NaCl)

FIGURE 4.34 Ocular irritation study by Chorioallantoic Membrane Test

a. CAM treated with Optimized formulation b. CAM treated with positive control (0.1 N

NaOH)

c. CAM treated with Negative control (0.9% NaCl)



4.10.2 Ocular Tolerability Study by Blinking index

he B.I. is defined as the ratio of the number of blinks (drug) divided by the number of blinks

(saline) and it is used as an indication of the drug irritability. The blinking index of saline solu-

tion and optimized formulation is 1.5 ± 0.4 and 2.4 ± 0.6 respectively shown in Table 4.38.

TABLE 4.38 Blinking index and clinical symptoms

Treatment Blinking Index

(Mean ± SEM)

Observation

Eye Closure (swelling)

Lacrimation Redness

Saline 1.5 ±0.4 0/4 1/4 0/4

Test formulation 2.4 ± 0.6 0/4 2/4 1/4

(n=4/treatment)

After topical application the strong correlation between the osmolarity of the solution and the

irritation / pain discomfort was observed. Obviously, other factors such as pH, the presence of

other chemicals (i.e. preservatives) and the drug's own chemical nature can greatly affect its

potential for eye discomfort / irritation.

4.10.3 Acute Ocular Irritation Study

Comparable scoring in symptoms like eye closure, lacrimation and redness with respect to sa-

line in rabbits indicates that the optimized formulation was well tolerated by the rabbits caus-

ing less discomfort as shown in Fig 4.35 and Table 4.38.

Saline after 60 min Optimized formulation after 60 min

FIGURE 4.35 Acute ocular irritation study



Thus the optimized formulation was found to be nonirritating with no ocular damage or ab-

normal clinical signs to the cornea, iris or conjunctiva observed. Hence the optimized formula-

tion was suitable for the eye instillation and viable alternative to conventional ocular formula-

tion.

4.10.4 Efficacy Study by Ovalbumin Induced Allergic Conjunctivitis Model

4.10.4.1 Edema scoring

Allergic conjunctivitis was induced as per protocol discussed in previous chapter.

34,

35 At 0.5

and 24 hr. after the instillations, the eye was challenged with ovalbumin solution (100 mg/ml,

30 µl). Edema was scored at 15, 30, 60, 90, and 120 min after the instillation of ovalbumin as

shown in Table 4.39. The edema scoring was done according to graded scale.

Following system was used for assigning the edema scores

0-No edema

1- Slight edema

2- Partial eversion of eye

3-Eyelid half-closed

4-Eye swelling, more than half eyelid closed

The observations which did not match exactly with the score mentioned in the following scor-

ing system were assigned a value between two adjacent scores up to 0.5. E.g. for scoring eyelid

edema a value of 1.5 was assigned in case of the score falling between 1 and 2.

TABLE 4.39 Edema scoring (At 0.5 h and 24 h after topical antigen challenge)

Group Treatment Sum of edema score (Mean ± SEM)

0.5 hr. 24 hr.

Non sensitized Saline + ovalbumin 0.00 ± 0.00 0.00 ± 0.00

Sensitized Saline + ovalbumin 17.66 ± 0.56 16.39 ± 0.02

Sensitized Ebastine (1% w/v) ocular formu-

lation + ovalbumin 3.56 ± 0.02* 9.43 ± 0.14*

Sensitized Ebastine (3 mg/kg) suspension in

0.5% CMC oral + ovalbumin 11.53 ± 0.16* 14.48 ± 0.14



*Significant compared to saline + ovalbumin treated sensitized animals, ANOVA followed by

Dunett‗s test, p<0.05. Each value represents mean ± SEM of 4 animals /treatment

FIGURE 4.36(a) Effect of optimized formulation on ovalbumin-induced conjunctivitis in

guinea pigs Onset of effect- Time point: 0.5 hr. *Significant compared to saline + Oval-

bumin treated animals, ANOVA followed by Dunett’s test, p<0.05. Each value represents

mean ± SEM.

FIGURE 4.36(b) Effect of optimized formulation on ovalbumin-induced conjunctivitis in

guinea pigsDuration of effect- Time point: 24 hr., *Significant compared to saline +

Ovalbumin treated animals, ANOVA followed by Dunett’s test, p<0.05. Each value rep-

resents mean ± SEM.



A B

C D

FIGURE 4.37 Edema response,

Instillation of ovalbumin in non sensitized animal do not induce edema, hence no eyelid

swelling (A). Severe edema, redness, and lacrimation were observed in sensitized animal

after ovalbumin challenge (B). ebastine (1% w/v) ocular formulation + ovalbumin (C).

ebastine (3 mg/kg) suspension oral + ovalbumin (D).

The Fig. 4.36 (a) and (b) indicate graphical presentation of scoring at 15, 30, 60, 90, and 120

min after topical antigen challenge at 0.5 hr. and 24 hr. Guinea pigs in the OA induced AC

model observed for clinical symptoms as shown in Fig.4.37. The results presented in Table

4.39 indicate that the optimized formulation (ebastine 1% w/v ocular) instilled 0.5 hr. and 24

hr. before the ovalbumin challenge caused significant inhibition of conjunctivitis symptoms.

While the oral ebastine caused significant inhibition of conjunctivitis symptoms at 0.5 hr. only.

It was also observed that compared to Ova challenge, optimized formulation showed 79.84%



inhibition at 0.5 hr., the effect persist up to 24 hr. with 42.46 % inhibition while oral ebastine

showed 34.71% inhibition at 0.5 hr. This result indicates that topical formulation of ebastine

showed better efficacy in ova induce conjunctivitis model at very low dose as compared to oral

4.10.4.2 Scratching Behavior

After the instillation of 30 μL /site of ovalbumin dissolved in normal saline solution (0.9%

NaCl) into the eye, guinea pigs were placed into the observation cage (1 animal/cage), and the

number of eye scratches was counted for 30 min. The scratching response was assessed after

topical antigen challenge at 0.5 hr. Animals treated with topical ebastine showed a significant

reduction in itch-scratch response as compared to ova challenge and oral ebastine as shown in

Fig 4.38.

FIGURE 4.38 Scratching response *Significant compared to saline + Ovalbumin treated

animals, ANOVA followed by Dunett‗s test, p<0.05. Each value represents mean ± SEM.

4.11 Histopathological Study

Histopathological conditions of conjunctiva after treatment with saline (negative control), sa-

line+ ovalbumin (positive control), and optimized formulation are shown in Fig. 4.39. No sig-

nificant damage/harmful or mild epithelium damage effects on the microscopic structure of the



conjunctiva treated with optimized formulation was observed in comparison to that of sample

treated with topical allergen ovalbumin indicating the safety of the optimized formulation for

ocular application. This was in accordance with the results obtained by Kyei et al. Conjunctiva

treated with topical allergen showed severe damage epithelium cellular layer and edema, as

well as neutrophil and eosinophil infiltration.

Saline (Negative Control)

Normal epithelium cell layer and no edema, as

well as neutrophil and eosinophil exudations in

the proper lamina.(40x)

Saline + Ovalbumin (positive control)

Damage epithelium cellular layer and edema, as

well as neutrophil and eosinophil infiltration.

(40x)

Optimized formulation (ebastine 1% w/v ocular formulation) + Ovalbumin

Mild epithelium damage and very less eosinophil infiltration than in conjunctiva of guinea pigs treat-

ed with ovalbumin alone. (40x)

FIGURE 4.39 Histopathological photomicrographs of the conjunctival tissues in ovalbu-

min induced allergic conjunctivitis in guinea pigs treated with (a) Saline (b) Saline +

Ovalbumin (c) Optimized formulation (ebastine 1% w/v ocular formulation) + Ovalbu-

min.



4.12 Pharmacokinetic Study

Single dose pharmacokinetic study was performed by measuring drug concentration in ocular

tissues. The blood and tissue samples collected from the rats were extracted, following the

method given in previous chapter. The analysis of the tissue samples were performed on

HPLC, according to the bioanalytical method of ebastine explained in previous chapter. The

pharmacokinetic parameters like Tmax, Cmax, and AUC were obtained by fitting the

experimental data to the pharmacokinetic model and explored using PK Solver software (Fig.

4.40). The formulation achieved peak drug concentration (Tmax) within 30 min. The Cmax

value was found 62.48 μg/g. The AUC of formulation was found 202.12 μg/gm*h. The higher

AUC of formulation may contribute to better clinical efficacy.

FIGURE 4.40 Fitting the experimental data to Pharmacokinetic Model (PK Solver 2.0)

Non-Compartmental Analysis of Ocular Tissue Data after Topical Input. Data represent

Mean Concentration (n=3/time point)

After single ocular instillation of 10 μl dose of 1% w/v ebastine ophthalmic formulation, the

concentrations in the ocular tissue were more than that in the plasma showing negligible sys-

temic absorption. These results are desirable for the ocular distribution of ebastine, because

0

10

20

30

40

50

60

70

80

0 0.5 1 2 3 4 8

Co

nce

ntr

atio

n (μ

g/g

)

Time (h)



high-level distribution of ebastine was observed in the ocular tissue, which is a target tissue for

pharmacologic effect (i.e. efficacy). The obtained pharmacokinetic parameters are summarized

in Table 4.40.

TABLE 4.40 Single dose pharmacokinetic study parameters

Sr. No.

Parameter Unit Value

1.

Time to reach peak Tissue

concentration

(Tmax )

h

0.5

2. Peak Tissue concentration

(Cmax) μg/gm 62.48

3. Area under the curve

(AUC 0-t) μg/gm*

h

202.12

4. Area under the curve

(AUC 0-inf_obs) μg/gm*

h

212.54

5. MRT 0-inf_obs h 2.78

The pharmacokinetic study conducted in rats revealed that the developed microemulsion gel

formulation of ebastine could retain at ocular tissue site up to 8 hr., which supports the in vivo

pharmacodynamics study conducted earlier. While systemic blood ebastine concentration after

ocular administration was extremely low or undetectable obviating concerns about systemic

toxicity. The developed formulation is successful in staying at the site of absorption for pro-

longed time thereby maintaining the drug concentration at tissue site, giving better topical

pharmacodynamic effect.



4.13 Stability Studies

4.13.1 Accelerated Stability Test by Centrifugation Stress Test

Stress stability study of the microemulsion sample was carried out by subjecting to centrifuga-

tion. A formulation shows no sign of phase separation when subjected to centrifugation at

9,000 rpm for 20 minutes. Thus, it was concluded that the microemulsion formulation was sta-

ble under stressful conditions.

4.13.2 Stability study as per ICH guidelines`

The stability studies of microemulsion and microemulsion based gel formulations were as-

sessed under various storage conditions as per ICH guidelines and the results are summarized

in Table 4.41 (a) and (b).



TABLE 4.41 (a) Stability study data for microemulsion formulation {*Data expressed as

mean ± SD (n = 3)}

Sampling

time (Month)

Visual assessment

pH Viscosity (cP)

at 25o

C

Globule size

(nm)

Zeta poten-

tial (meV)

0 No phase separation 6.9 ± 0.12 13.19 ± 0.121 142 ± 0.16 -22.6 ± 0.39

At room temperature (25o

C ± 2o

C / 60% RH ± 5% RH)




6 No phase separation 6.4 ± 0.17 15.09 ± 0.001 148 ± 0.12 -22.0 ±0.89

At refrigeration condition (2-8oC)




6 No phase separation 6.8 ± 0.31 14.09± 0.224 147 ± 0.56 -25.7 ± 0.34

At 40oC ± 2

oC / 75% RH ± 5% RH




6 No phase separation 7.1± 0.19 13.29 ± 0.249 145 ± 0.04 -24.0 ± 0.06



TABLE 4.41 (b) Stability study data for microemulsion based gel formulation {*Data

expressed as mean ± SD (n = 3)}

Sampling

time (Month)

pH Viscosity

(cP) at 25oC, 100 γ (s-1)

Drug content

(%)

0 6.8 ± 0.44 1611.1 ± 20.14 98.22 ± 0.40

At room temperature (250C ± 2

0C / 60% RH ± 5% RH)

1 6.7 ± 0.09 1629.1 ± 18.19 98.22 ± 0.40

2 6.9 ± 0.51 1591.1 ± 17.12 97.13 ± 0.24

3 7.0 ± 0.87 1607.1 ± 14.19 96.56 ± 0.78

At refrigeration condition (2-80C)

1 6.4 ± 0.67 1705.1 ± 12.34 97.42 ± 0.56

2 6.6 ± 0.87 1639.1 ± 21.19 95.67 ± 0.89

3 6.3 ± 0.43 1714.1 ± 19.11 95.89 ± 0.01

At 400C ± 2

0C / 75% RH ± 5% RH

1 6.6 ± 0.02 1411.1 ± 13.13 97.67 ± 0.21

2 6.3 ± 0.89 1311.1 ± 10.18 96.91 ± 0.67

3 6.2 ± 0.30 1211.1 ± 17.17 96.97 ± 0.01



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CHAPTER 5 Summary and Conclusion


CHAPTER 5

Summary and Conclusion



5.1 Summary of the Work

Allergy is not a disease itself, but a mechanism leading to disease. Ocular allergy is wide-

spread and growing healthcare problem that has a significant negative impact on quality of life

both in adults and children. A major problem in ocular therapeutics is the attainment of an op-

timal drug concentration at the site of action. Poor bioavailability of drugs from ocular dosage

forms is mainly due to the precorneal loss factors which include tear dynamics, non-

productive absorption, transient residence time in the cul-de-sac and relative impermeability of

the corneal epithelial membrane. Oral antihistamines cause unfavorable effects on heart like

QT prolongation, severe gastric distress, decreased tear production, resulting in dryness of the

ocular surface, which exacerbates ocular discomfort and increasing susceptibility of eye to ir-

ritation. Topical antihistamines are preferred for treating ocular allergies over oral agents since

their direct application at the site of action results in rapid onset and superior efficacy with less

systemic side effects. Hence, topical formulation was developed to achieve onsite exposure of

ebastine for ocular allergies.

The procured drug sample was visually observed for its color and was compared with the re-

ported appearance of the drug. Melting point is one of the identification test method for organ-

ic substances. Hence, it was determined for the sample by capillary method using melting

point apparatus (VMP-D, Veego). The IR spectroscopy was conducted using an FTIR spectro-

photometer and the spectrum was recorded in the wavelength region of 4000– 400 cm−1

IR

spectroscopy and DSC study of pure ebastine and physical mixtures with excipients were con-

ducted using an FTIR spectrophotometer (Bruker Alpha-one, Bruker Optik, Germany) and

DSC analyzer (Pyris-1 DSC, PerkinElmer) in order to detect the existence of a possible inter-

action between drug and excipients. IR study revealed that there is no interaction between

drug and excipients as all characteristic peaks of pure ebastine were found in the physical mix-

ture. The DSC thermogram of the pure ebastine and physical mixture exhibited the character-

istic endothermic peaks at 88.69 °C and 87.50 °C respectively, indicating the absence of inter-

action between the drugs thereby proving drug-excipient compatibility. UV analytical method

was used for determination of drug content, % drug release and stability samples. HPLC ana-

lytical methods were developed and validated for estimation of ebastine in ocular tissue.



The solubility of ebastine was determined in various oils, surfactants and cosurfactants. Drug

powder was added in excess to each of the oils, surfactants and cosurfactants, thereafter sub-

jected to vortexing. After vortexing, the samples were kept for 24 h at ambient temperature for

attaining equilibrium. The equilibrated samples were then centrifuged at 3000 rpm for 20 min

to remove the undissolved drug. The aliquots of supernatant were filtered through 0.45 μm

membrane filters and solubility of ebastine was determined by analyzing the filtrate spectro-

photometrically (UV1800, Shimadzu) after dilution with methanol at 252 nm. Campul MCM

EP was selected as the oil phase. The blend of Labrasol with Tween 80 and blend of Propylene

glycol with glycerol were selected as surfactant and co surfactant, respectively. Double dis-

tilled water was used as an aqueous phase. Pseudo ternary phase diagrams were constructed

using aqua- titration method at ambient temperature (25°C) by Prosim software. Three phase

diagrams were obtained for three different Smix individual ratios 1:1, 2:1, and 3:1. The com-

paratively maximum microemulsion area was obtained in 2:1 Smix ratio. The selected Smix

ratio was further studied by Smix blend, 2(1:1):1, 2(1:1): 1(1:1).

D-optimal mixture design (Design-Expert 7.0.0 (Stat-Ease Inc., Minneapolis, USA) was se-

lected because the generalized variance of the estimates of the coefficients is minimized. Dif-

ferent design constraints, i.e. A (amount of oil), B (amount of Smix), and C (amount of water)

were taken at high and low levels. The sum of A, B, and C were kept fixed at 100%. The ef-

fect of these formulation variables was studied on response variables like % Transmittance,

globule size and viscosity. ANOVA was applied to determine the significance and the magni-

tude of the effects of the variables and their interactions. The probability value (α) for deter-

mination of statistical significance was set at 0.05, which indicated that a ―hypothesis‖ theory

would be rejected if their corresponding p-values were ≤ 0.05. Models were selected on the

basis of sequential comparison and lack of fit test.

Response surface, contour plot, residual plot and overlay plots were constructed for the re-

sponse variables. It was observed from the response variables plots of Globule size that as the

concentration of oil increases, globule size also increases while the concentration of Smix in-

crease then globule size decreases. It was observed from the response variables plots of vis-



cosity that as the concentration of Smix increases and decreases amount of water, viscosity

increases. It was observed from the response variables plots of % transmittance that as the

concentration of oil increases % transmittance decreases and as Smix increases % transmit-

tance increases. Further, linear correlation was found analogous for actual response and predi-

cated response. The reliability of these response surfaces was also confirmed by the corre-

sponding residual plot between the experimental run and the internally studentized residuals

for all response variables. These findings revealed that all points fall within a confidence in-

terval of 95%. Experimental validation of DOE trials for formulation variables was undertaken

by formulation and characterization of microemulsion formulation at the check point batch

suggested by the software. The observed values of globule size (nm), viscosity (cps) and

transmittance (%) were comparable with the predicted values of same responses establishing

the reliability of the optimization procedure. Calculated percentage prediction error was found

to be less than 5 percent, confirming the validity of D- optimal mixture design for microemul-

sion formulation optimization.

Resultant developed formulation shows droplet size (142 ± 0.16 nm), Polydispersity Index

(below 1), refractive index (1.369 ± 0.04). The pH value of the developed formulation was 6.9

± 0.12, which can be easily buffered by tear fluid (pH 7.2-7.4), consequently, it is adequate to

apply to the eye without causing irritation, reflex tear and rapid tear blinking. An Osmolarity

of developed formulation was found to be 291±0.301mOsm/L. Low microemulsion surface

tension ensures good spreading effect on ocular surface and mixing with precorneal film com-

ponents, thereby improving contact with ocular surface. The surface tension of the developed

formulation was found to be 34.75 ±0.13 mN/m. Zeta potential and viscosity of developed

formulations was found to be -22.6 ± 0.39 mV and 13.19 ± 0.121cps respectively. The per-

centage of drug content of optimized formulations was found to be 97.09 ± 0.12%. The devel-

oped microemulsion was found in the limit of acceptable droplet size range for ocular use and

presented physical stability. Physicochemical parameters like pH, osmolarity, surface tension

were found in the range which favors its ophthalmic suitability. Microstructures of micro-

emulsion was studied by transmission electron microscopy, it directly produces high-

resolution images. It can capture any co-existent structure and microstructural transitions, per-

formed using Technai-20, Phillips, Holland, Electron source: LaB6, Tungsten Filament. The



morphology of the droplets of optimized formulation measured using TEM showed spherical

shape and uniform droplet size of optimized microemulsion. Because the loaded ebastine mi-

croemulsion globules are nanometric and morphologically spherical, they are not expected to

cause ocular irritation. Further, the optimized microemulsion formulation was sterilized using

membrane filtration unit by passing the formulation through 0.22 μm membrane filter under

aseptic conditions. The optimized formulation was found to pass the sterility test carried out

using fluid thioglycolate media and soyabean casein digest media to detect aerobic, anaerobic

bacteria and fungal organisms respectively.

The optimized microemulsion was converted into microemulsion based gel. The addition of

the gelling agent increased the viscosity in comparison to parent microemulsion. Different gel-

ling and mucoadhesive agents were screened based on desired viscosity. Carboxy Methyl Cel-

lulose and Sodium hyaluronate were selected as polymers based on literature. Based on pre-

liminary trial, 1% CMC and 1.5% SH polymers dispersion was found satisfactory to get opti-

mum viscosity. The dispersion was formed by suspending the polymers in water. The polymer

dispersion kept for overnight to form viscous gel matrix. Prepared microemulsion and polymer

dispersion was mixed in 1:1 w/w ratio. Smooth viscous, transparent gel was formed. The un-

derlying procedure for preparation of microemulsion based gel was carried out strictly in asep-

tic area to maintain the sterility of overall formulation.

The microemulsion based gel was evaluated for physical examinations like homogeneity, con-

sistency, texture, etc. Gel was also evaluated for appearance, pH, rheology, drug content, mu-

coadhesive strength (determined by modified two-pan balance method) and spreadability (de-

termined by taking 0.5 g gel between two cellophane membranes and placing 100 g weight on

it for 1 minute. The diameter of the area in which the gel got spread was measured). The rheo-

logical analysis was done by means of Plate and cone Viscometer at different shear stress. The

pH value of the 1% w/v aqueous solution of the prepared microemulsion based gel was found

to be is 6.8 ± 0.44. The percentage of drug content was found to be 98.22 ± 0.40 %. The mu-

coadhesive strength and spreadability of the gel was found to be 2.8 cm/gm gel and 15,401.7

dynes/cm2 respectively. Proper spread of the gels on the ocular surface will ensure increased

absorption of the drug after ocular administration. The pseudo plastic character of precorneal



tear film should be disturbed less/or not disturbed by the administration of ophthalmic prod-

ucts. The ocular shear rate is about 0.03 s-1

during interblinking periods and 4250 – 2850 s-1

during blinking. The viscoelastic fluid having high viscosity under low shear rates and low

viscosity under high shear rates, called as pseudo plastic fluid, is often preferred for ophthal-

mic application. The Rheogram of microemulsion gel and microemulsion gel diluted with tear

fluid (To mimic physiological condition, formulations were mixed with artificial tear fluid

(ATF) in a ratio of 40:7) exhibited pseudo-plastic behavior, i.e., decrease in the viscosity with

increase in angular velocity exhibiting its suitability for ophthalmic use.

The in vitro drug release study was carried out using the dialysis bag method. Higher % ebas-

tine released from microemulsion (89.19 ± 2.45%) was found as compared to microemulsion

based gel (71.34 ± 2.34%) within 8 hr. However, microemulsion gel was able to sustain the

release of the remaining ebastine for up to 24 h. This might be due to possible matrix effect on

release of ebastine due to incorporation of microemulsion in CMC and SH gel, a micro gel

layer forms around the droplets that can hinder drug diffusion from the oil phase, so the rate

and the amount of the released drug may decrease, while the release rate of the drug from mi-

croemulsion depends on the rate of diffusion of the drug from oil droplets. The possibility of

the drug partition between the oil and the water phases in the presence of the surfactant posi-

tioned at the oil–water interface prior to release. Formulation provided the highest in vitro

drug release with the ability of providing a sustained release over 24 hr., thus reducing fre-

quency of application and improving patient compliance.

For In-vivo study, the animal experimental protocol was approved by the Institutional Animal

Ethics Committee (IAEC) Reference No. 984/01/2017-07 for the use of animals in the study.

Utmost care was taken to ensure that animals were treated in the most human and ethically

acceptable manner. The ocular potential of optimized ocular formulation assessed by perform-

ing in vitro study like hen's egg chorioallantoic membrane test (HET-CAM) and blinking in-

dex for tolerability and in vivo antiallergic efficacy study in ovalbumin (OA)-induced allergic

conjunctivitis (AC) in guinea pigs followed by histopathology. After sensitization period, an-

imals were used for the experiments assessing efficacy of formulation. The optimized formu-

lation (20μL), saline, (20μL) was instilled into the right eye of respective group and for oral;



ebastine (3 mg/kg) in 0.5% CMC was given. At 0.5 and 24 hr. after the instillations, the eye

was challenged with ovalbumin solution (100 mg/ml, 30μl). Edema was scored at 15, 30, 60,

90, and 120 min after the instillation of ovalbumin. For evaluation of edema, scoring system

was used. In the same sensitization protocol, eye scratching behavior and edema were scored

at periodic interval after the topical antigen challenge (instillation of ovalbumin) followed by

histopathology. Eye scratching behavior was defined as fore-limb movements over two times

directed to the ocular surface. The results shown that the optimized formulation instilled 0.5

hr. and 24 hr. before the ovalbumin challenge caused significant inhibition of conjunctivitis

symptoms. While the oral ebastine caused significant inhibition of conjunctivitis symptoms at

0.5 hr. only. It was also observed that compared to ova challenge, test formulation showed

79.84% inhibition at 0.5 hr., the effect persisted up to 24 hr. with 42.46% inhibition while oral

ebastine showed 34.71% inhibition at 0.5 hr. This result indicates that topical formulation of

ebastine showed better efficacy in ova induce conjunctivitis model at very low dose as com-

pared to oral. Further, the study indicated that said formulation has a quick onset and the dura-

tion of effect sufficient to provide relief from symptoms for 24 hr.

Ocular irritation studies by HET-CAM assay showed that the developed formulation does not

cause any irritation to the blood vessels. Acute ocular irritation test was performed using rab-

bits; the animals were observed up to 60 min for redness, swelling, watering of the eye and

results showed that developed formulation was non-irritant to the eye. In histopathological

evaluation, ocular formulation caused mild epithelium damage with less eosinophil infiltration

than in conjunctiva treated with ovalbumin alone. Data obtained in different groups expressed

as mean ± Standard error of mean (SEM) were analyzed using one-way ANOVA followed by

Dunett's test. Statistical significance was considered as p value < 0.05. Statistical analysis was

performed using the Sigma Stat, Version 3.1. (SPSS Inc., USA)

Further, the optimized formulation was subjected to pharmacokinetic study. Single dose

pharmacokinetic study was performed by measuring drug concentration in ocular tissues. The

formulation achieved peak drug concentration (Tmax) within 30 min. The Cmax value was

found 62.48 μg/ml. The AUC of formulation was found 202.12 μg/ml*h. The higher AUC of

formulation may contribute to better clinical efficacy. After single ocular instillation of 10 μL



dose of 1% w/v ebastine ophthalmic formulation, the concentrations in the ocular tissue were

higher than that in the plasma showing negligible or no systemic absorption. These results are

desirable for the ocular distribution of ebastine, because high-level distribution of ebastine was

observed in the ocular tissue, which is a target tissue for pharmacologic effect (i.e., efficacy).

Stress stability study of the microemulsion formulation was carried out by subjecting it to cen-

trifugation. The stability of the microemulsion and microemulsion based gel were assessed

under different storage conditions as per ICH guidelines, at room temperature (250C ± 2

0C /

60% RH ± 5% RH), at refrigeration condition (2-80C), at 40

0C ± 2

0C / 75% RH ± 5%RH. No

significant changes were observed during the stability studies in measured parameters, while

the microemulsion gel shown slight change in viscosity under accelerated temperature and

humidity condition, hence recommended condition for proper storage for developed formula-

tion is at room temperature and /or refrigeration.

5.2 Achievement with Respect to the Objective

Topical ocular therapy could prove to be superior to systemic therapy in treating ocular aller-

gies. Hence, topical formulation was successfully developed to achieve onsite exposure of

ebastine for ocular allergies. The use of surfactant –cosurfactant blend system resulted in for-

mulation of drug delivery system with low surfactant level. The design expert allowed optimi-

zation of formulation and response variables as per ocular site application requisite. By the use

of gelling and mucoadhesive polymer, the formulation residential time was successfully in-

creased and the drug released for prolong period. The ocular presentation of model drug

through micro emulsified form shown tolerability and efficacy as well as increased bioavaila-

bility due to site specificity. The analytical method was successfully developed and validated

for the estimation of ebastine in ocular tissues.

5.3 Conclusion

With the present investigations, it may be concluded that microemulsion of a poorly soluble

drug ebastine was successfully formulated and optimized using the systematic approach of de-

sign of experiments (DOE) by water titration method. Studies of equilibrium solubility were

conducted in different oils, surfactants and co- surfactants to rationally optimize the formula-



tion using D- optimum mixture design. The developed microemulsion was found in the limit

of acceptable droplet size range for ocular use and presented physical stability. Physicochemi-

cal parameters like pH, osmolarity, isotonocity were found in the range which favors its oph-

thalmic suitability.

Mucoadhesive gel was prepared with the optimized microemulsion. The addition of the gel-

ling agent increased the viscosity in comparison to parent microemulsion. The results of the

release study indicated that formulation prolonged the precorneal retention owing to mucoad-

hesion by polymer. Hence, bioavailability at the site of action of said drug was found to be

significantly increased. Further, formulation also proved its tolerability (no signs of irritation,

bleeding, vessel lysis and coagulation) in vitro HET-CAM assay by preserving normal archi-

tecture of blood vessels and in vivo acute irritation study showed reduced lacrimation, blinking

index and redness. The in vivo efficacy study proved that the formulation to be superior in

treating allergic conjunctivitis by exhibiting statistically significant reduction in conjunctivitis

symptoms like edema and scratching as compared to oral ebastine. In histopathological evalu-

ation, ocular formulation caused mild epithelium damage with less eosinophil infiltration than

in conjunctiva treated with ovalbumin alone.

In a nutshell, the microemulsion based gel formulation developed using the design expert ap-

proach held great potential as a possible alternative to traditional oral formulations of poorly

soluble ebastine to improve solubility and bioavailability due to site specificity as well, fitting

ocular application prerequisite. These data from in vitro, pharmacodynamics, nonclinical stud-

ies indicate effective topical delivery of ebastine to desired target tissues along with a favora-

ble safety profile, making 1 % w/v ebastine ophthalmic microemulsion based gel a promising

treatment for allergic conjunctivitis. These findings further warrant clinical investigation.

Appendices


Appendix I

Approval Certificate from CPCSEA & IAEC Committee for Animal Study

Appendices


Appendix II

Dose Calculation Method

Molecular Weight of Ebastine: 469.7gm/mol

4697 µg-1000 mL =1 µM,

4697 ng -1000 mL = 1 nM,

Therefore, 0.4697 ng/ml = 1 nM,

H1 histamine antagonist, IC50 (Antihistaminic effect) = 45 nM

Therefore, concentration required to give effect is 0.470 x 45 nM = 21.13 ng/mL

Inhibit T cell proliferation and the production of Th2-type pro-inflammatory

cytokines by macrophages, IC50= 10 μM

Therefore, concentration required to give effect is 469.7 x 10= 4697 ng/mL

Dose instilled is 10 µL of 1% w/v optimized formulation containing 100 µg of drug.

Thus, as per the pharmacokinetic data, significant tissue concentration was observed up to 8 hr.

of dosing. The study revealed that ebastine concentration in targeted ocular tissue is >>>> IC 50

(oral) values to inhibit H1 histamine antagonist and inhibition of T cell proliferation, the

production of Th2-type pro-inflammatory cytokines by macrophages.


List of Publications

Publications from Research Work

A research article titled ―Rationalized Approach for Formulation and Optimization of

Ebastine Microemulsion Using Design Expert for Solubility Enhancement‖ Mehetre J,

Vimal K, Mehta T, Gohel M, Surti N, Journal of Drug Delivery and Therapeutics, 2019;

9(3-s):386-397.

A research article titled ―Ocular Tolerability and Efficacy of Ebastine Colloidal

Formulation in Allergic Conjunctivitis‖ Mehetre J, Vimal K, Mehta T, Gohel M, Surti N,

International Journal of Pharmaceutical Sciences and Drug Research, 2019; 11(4): 129-

136.

A Colloidal Drug Delivery System for Antiallergic Drug

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