“Design

“Design, Development and Characterization of Ophthalmic Emulsion of Immunosuppressant Drug Formulation Using Microfluidizer as a Technology.”
A Thesis Submitted to
NIRMA UNIVERSITY
In Partial Fulfilment for the award of the degree of
MASTER OF PHARMACY
IN
PHARMACEUTICAL TECHNOLOGY & BIO-THERAPEUTICS
BY
Harshil Shah, (16MPH105) M.PHARM.

Under the Guidance of
Dr. Subhash Gore – GUIDE
Vice-President, Pharma Research, Lupin Limited.

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Dr. Renuka Mishra – CO-GUIDE
Assistant Professor, Institute of Pharmacy, Nirma University.

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CERTIFICATE
This is to certify that the dissertation work entitled “Design, Development and Characterization of Ophthalmic Emulsion of Immunosuppressant Drug Formulation Using Microfluidizer as a Technology” submitted by Mr.Harshil Shah with Regn. No. (16MPH105) in partial fulfilment for the award of Master of Pharmacy in “Pharmaceutical Technology and Bio pharmaceutics” is a bonafide research work carried out by the candidate at the Department of Pharmaceutics, Institute of Pharmacy, Nirma University and at Lupin Research Park, Pune. Under our guidance. This work is original and has not been submitted in part or full for any other degree or diploma to this or any other university or institution.

Industrial/Hospital Guide Academic Guide:
Dr.Subhash Gore
Vice President
Pharma Research
Lupin Research Park, Pune.

Dr. Renuka Mishra
M.Pharm., Ph.D.

Assistant Professor,
Department of Pharmaceutics,
Institute of Pharmacy,
Nirma University
Prof. Tejal Mehta
M. Pharm., Ph.D.

Professor & Head,
Department of Pharmaceutics,
Institute of Pharmacy,
Nirma University
Prof. Manjunath Ghate
M. Pharm., Ph.D.

Director
Institute of Pharmacy,
Nirma University

Date, Month, 2018

DECLARATION
I hereby declare that the dissertation entitled “Design, Development and Characterization of Ophthalmic Emulsion of Immunosuppressant Drug Formulation Using Microfluidizer as a Technology.” is based on original work carried out by me under the guidance of Dr.Subhash Gore, Vice President, Lupin Research Park and Dr.Renuka Mishra, Assistant Professor, Department of Pharmaceutics, Nirma University. I also affirm that this work is original and has not been submitted in part or full for any other degree or diploma to this or any other university or institution.

Mr. Harshil Shah (16mph105)
Department of Pharmaceutics,
Institute of Pharmacy,
Nirma University,
Sarkhej-Ghandhinagar Highway,
Ahmedabad-382481;
Gujarat, India.

Acknowledgements
It gives me immense pleasure today when I take an opportunity to acknowledge all those personalities who contributed directly or indirectly to my project. This research would not have been possible without the whole hearted encouragement, guidance, support and cooperation of my beloved family, teachers, friends, well wishers and relatives. Probably I would never have achieved this without their support and blessings. With profound appreciation, I acknowledge to one and all I take this opportunity to proudly place on record my profound sense of gratitude to Dr. Subhash Gore, Vice President of Lupin Research Park, and Pune for his valuable guidance, encouragement and continued support throughout the course of this work.

I equally thankful to Dr. Tejal A. Mehta Associate Professor, Head, Dept. of Pharmaceutics, Institute of Pharmacy, Nirma University for her unending encouragement, friendly nature, timely suggestions and total understanding. With profound pleasure, I express my deepest gratitude to my esteemed guide, Dr. Renuka Mishra Assistant Professor, Department of Pharmaceutics and Pharmaceutical Technology, Institute of Pharmacy, Nirma University for her erudite guidance, timely suggestions, continuous encouragement and critical remarks for the entire span of my work. I look her as my ideal and treasure, the relationship with her as a student and a human being now and forever in future.

I am thankful to Dr. Vinayak Kadam, Sr. Research Scientist of Lupin Research Park, Pune, Radheshyam Kale, Sunil Londhe, Dharmaraj More, Rahul Patil and Prashant Dolas.
I am deeply indebted and thankful to Dr. Shital Butani, Dr.Jigar Shah, Dr. Mayur Patel and Dr. Dhaivat Parikh and for their keen interest and valuable support. They were always available to give me the suggestions.

A special word of gratitude to my friends who were always there besides me with their hand of support and encouragement to make my effort a successful task.

I would also like to acknowledge my colleagues Aishwarya Mishra, Ankita Singh, Dhruvi Solanki,Dhwani Desai, Jagat Maniyar,Kartik Hariharan,Nidhi Patel, Nidhish Patel, Niharika Devli,Nimesh Patel,Rasesh Patel,Riddhi Patel,Shivangi Kaul,Smit Patel,Viraj Makwana for their help throughout my work.

Last but not the least, I am indebted infinitely to love, care, patience and trust being showered on me by my family-My Father Mukeshbhai Shah, My Mother Belaben Shah. With their consistent prayers, affectionate blessings, selfless care and endless confidence in me.I would have never come to this stage of writing this acknowledgement.

Harshil Shah

Abbreviations
API = Active Pharmaceutical Ingredients
ANDA =Abbreviated New Drug Application
Mg =Milli gram
IP=Indian Pharmacopoeia
USP =United Status Pharmacopoeia
?c =Degree Centigrade
?F =Degree Fahrenheit
RPM =Revolutions per Minute
W/v =Weight per Volume
HDPE=High Density Polyethylene
LDPE=Low Density Polyethylene
RH=Relative Humidity
QBD=Quality by Design
PH=Potential of Hydrogen
USM= Up Stream Mixer
CPS=Centipoise
PDI= Polydispersivity Index
Mv= Milli volt
BCS=Bio-Pharmaceutical Classification System
Con.=Concentration
Avg.=Average
List of Figures
Sr No. Title Page No
1 Anatomy of Eye 10
2 Flow of Tears 12
3 Anatomy of Tear Film 13
4 Ophthalmic Drug Delivery System 14
4 Factors Affecting Intraocular Bioavailability 14
5 Advantages of Ocular Drug Delievery 14
6 Disadvantages of Ocular Drug Delievery 15
7 Difference Between Normal Eye and Dry Eye 19
8 Microfluidizer Process 28
9 Design of Formulation 34

List of Tables
Sr
No. Title Page No
1 Anatomy of Tear System 12
2 Physical Properties of Tear Film 13
3 Three Layers of Eye Tear Film 13
4 Approaches To Improve Ophthalmic Drug Delivery 16
5 Classification of Dry Eye Disease 19
6 Difference Between Healthy Eye and Dry Eye 20
7 Prevalence of Dry Eye in U.S 22
8 Prevalence by Age and Sex 23
9 Marketed Products of Dry Eye Disease 23
10 Various Properties of Drug Substance 24
12 List of Excipients Used in formulation & Development 24
13 List of Equipments Used in formulation & Development 29
14 Drug Substance Solubility in Various Media 31
15 Reference Product Characterization 32
16 Stressed Conditions & total impurity(%) of forced degradation study 33
16 Drug-Excipient Compatibility Study 33
17 Container Closure Study for Measurement of Assay 34
18 Container Closure Study for Measurement of Related Substance 34
18 Critical Process Parameters 35
19 Formulation of Trial 1,4,5 36
20 Formulation of Trial 2 37
21 Formulation of Trial 3 38
22 Formulation of Trial 6,7,8,9,10 40
23 Formulation of optimized Trial 49
24 Stability Study of optimized Trial 51
25 Variables of Formulation Trial 6 to 10 52
TABLE OF CONTENTS
Chapter No. Title Page No.

Abstract 9
1.0 Introduction 13
1.1 Anatomy of Eye and Tear Film 14
1.2 Ophthalmic drug delivery system 15
1.3 Formulation Approaches to Improve Ocular Delivery 16
1.4 Disease Profile 19
1.4.1 Introduction of Disease 20
1.4.2 Causes and Complications of Disease 20
1.4.3 Pathophysiology of Disease 21
1.4.4 Sign & Symptoms 21
1.4.5 Diagnosis & Treatment 21
1.4.6 Current Scenario of Dry Eye Disease 22
1.4.7 Marketed Products for Ophthalmic Drug Delivery 22
2.0 Aim & Objective 24
3.0 Drug and Excipient Profile 24
3.1 Introduction to Drug Substance X 24
3.2 Excipient Profile 26
4.0 Literature Review 26
4.1 Literature Review on Dose and Technology specific
4.2 Literature Review on Drug and Excipient specific
4.3 Literature Review on Formulation specific
4.4 Literature review on Patent 25
26
27
28
5.0 Materials and Methods 29
5.1 Material and Equipments
5.3 Identification of Drug
5.4 Estimation of API 29
29
30
6.0 Methodology 30
7.0 Experimental Work 31
7.1 Preformulation study 31
7.1.1 Drug Solubility Data 31
7.1.2 Innovator Product Characterization 31
7.1.3 Drug-Excipient Compatibility Study 32
7.1.4 Container Closure Study 33
7.2 Formulation Strategy 34
7.2.1 Design OF Formulation 34
7.2.2 Critical Process Parameters 34
7.4 Preliminary Trials using QBD Approach 36
7.5 Optimized Formulation 48
7.6 Stability Study 50
8.0 Summary 55
9.0 References 56

ABSTRACT
Delivery of drugs by using unadventurous drug delivery such as solutions is producing a challenge to the treatment of ocular diseases like Dry eye disease. The Chief Hurdles for this type of drug delievery are less corneal contact time, lachrymal fluid secretion and various barriers like lachrymal fluid-eye barriers and blood-ocular barriers. A number of formulations have been made to improve the penetration of drug and extend the contact time of drug when it applied topically into eye. By using Microemulsion as a potential drug carrier for ocular drug delivery possess various properties like remarkable thermodynamic stability, very low surface tension, lesser viscosity, smaller globule size which ultimately improved ocular drug delivery into eye plus it possess both hydrophilic as well as lipophilic characteristics.This Research shows the microemulsion which is prepared by using Microfluidizer is categorized for various properties like PH,Viscosity,Globule Size and stability studies. The Reported Results shows enriched patient compliance matched to unadventurous eye drops and most importantly enhance the ocular penetration of drugs in tissues plus possess high corneal interaction period. Nevertheless, the enhancement in field there is still need of emerging a nanoemulsion which could touch the target site and retain Non-intrusive mode of drug administration.

Key Words: – Ocular Drug Delivery, Anatomy of Tear Film, Challenges to Ocular Delivery, Ophthalmic Emulsion, Microfluidizer.

Chapter 1.0
INTRODUCTION
1.1 Anatomy of Eye and Tear Film:-
Anatomy of Eye:-
The human eye, it is the organ responsible for the senses of light, It is a very complex structure.
Human Eye contains several parts which are shown in figure:-

Figure SEQ Figure * ARABIC 1 Anatomy of Eye

1) Sclera
It is the superficial layer of the eye.

It is the white part of Eye and Muscles which are liable to moving the eye are attached to eye at sclera.

2) Cornea
It is the Opaque and dome shaped part of eye. Light Rays which are coming from the outside 1ST passes through cornea before reaching the lens.

It doesn’t contain blood vessels and it is extremely sensitive to pain.

3) Choroid
Internal Layer of eyeball which is located between sclera and retina.

Its provide nutrients and oxygen to exterior surface of retina.

4) Anterior Chamber
Space between Cornea and Sclera is recognized as anterior chamber.

It is filled with fluids which is known as Aqueous Humour.

It is also entitled as Anterior Cavity.

5) Posterior Chamber
It is superior than Anterior Chamber.

It is filled with fluid called as Vitreous Chamber.

6) Iris
The Choroid continuous at front of eyeball to form iris.

It Contains Circular Muscles and Radial Muscles.

When Circular Muscle contracts it makes pupils smaller and When Radial Muscle Contracts it makes Pupils wider.

It regulates the amount of light which enters into eye.

7) Lens
It is biconvex transparent disc made up of proteins.

It is located behind the iris and focuses light on the retina.

8) Retina
It is the innermost layer of eyeball.

It contains Photoreceptors which senses lights light.

These Photoreceptors contains Rodes and Cones.

Cones allows us to detect color and Rodes allows to see in poor light.

9) Optic Nerve
It located at the behind of eyeball.

It transmits signals from retina to brain by ganglionic cell which is the nerve cell of retina.

10) Conjuctiva
It lies inside the eyelids and covers the sclera.

It keeps the anterior surface of eye wet and lubricated.

Anatomical And Physiological Considerations in Topical Delivery
PRECORNEAL AREA
Precorneal Tear Film
Good visual function and corneal transparency require a uniform dry surface. This is accomplished by the tear film, which covers and lubricates the cornea and External Globe. The trilaminar structure of the tear film is shown in figure. Attached to the glycocalix of Corneal/Conjuctival Surface is a mucous layer, which consists mainly glycoproteins. It plays an important role in stability of tear film as well as in wetting of Corneal and Conjuctival Epithelium. The Middle aqueous layer institutes about 98% of tear film. It is composed of water, electrolytes and various proteins. The outer lipid layer prevents the evaporation of tear fluid. It consists of triacylglycerol and Phospholipids and is spread over the aqueous layer during blinking.

Figure 3 Anatomy of Tear Film
Nasolacrimal drainage system
The lacrimal gland which is responsible for tear fluid excretion. Secreted fluid is spread over surface of cornea during blinking and ends up in puncta when upper eye lid approaches to lower lid. The Process of blinking creates a suction mechanism which results in tears flowing through lacrimal canaliculi into lacrimal sac. Fluid from lacrimal sac then goes into nasolacrimal duct, which empties into inferior nasal passage. This Passage is highly vascular area and responsible for most of the drug absorption and subsequent side effect of topically administered drug.

The cul-de-sac normally holds 7-9µL of tear fluid, with normal tear flow rate being 1.2-1.5µL/min.Loss from the Precorneal area by drainage, tear fluid turnover and noncorneal absorption plays an important role in determining of ocular bioavailability of a drug.As the drainage rate is much faster than ocular absorption rate, most of topically applied drug is eliminated from Precorneal area within the first time. Tear production can be divided into basal, reflex and emotional tearing.

Flow of Tears:-

Table SEQ Table * ARABIC 1 Anatomy of Tear System

Figure SEQ Figure * ARABIC 2 Flow of Tears
Physical Properties of Tear Film:-
Table SEQ Table * ARABIC 2 Physical Properties of Tear Film
Properties Value
H2O 98.4%
Solids 1.9%
pH 6.6-7.6
Osmolality 300-310 mosm/l
RI 1.39
Tear Evaporation 0.15µl/min in 30% humidity
Surface Tension 40 dynes/cm

1.2 Ophthalmic Drug Delivery System:-
Administration of Drug into eye is primary requirement to treat ophthalmic diseases.

Eye is most nearby site for topical administration of Drug.

Ideal Ophthalmic Drug Delivery must be capable to endure the drug release and remain in locality in prolong period of time.

Figure:-Ophthalmic Drug Delivery System
Factors Affecting Intraocular Bioavailability:-

Figure SEQ Figure * ARABIC 4 Factors Affecting Bioavailability
Ophthalmic Dosage Forms:-
Ophthalmic Dosage Forms are sterile dosage forms free from foreign Particles, Fittingly Compounded and Packaged for installed into the eye.
Advantages of Ocular Drug Delivery:-

Figure 5 Advantages of Ocular Drug Delivery
Disadvantages of Ocular Drug Delivery:-

Figure 6 Disadvantages of Ocular Delivery
1.3 Approaches To Improve Ophthalmic Drug Delivery:-
Approaches to improve penetration in eye have been done to increase penetration in eye and Prolong the contact time in eye.

Conventional Drug Delivery Systems:-
Conventional dosage forms such as solutions, suspensions and ointments description for nearly 80% of currently accessible ophthalmic formulations in the market. They produce easiness of administration and low production costs. There are also disadvantages which includes very short contact time with the ocular surface and faster nasolacrimal drainage both leads to poor penetration of drug into eye.

Polymeric Delivery Systems:-
Polymer drug delivery used for ocular drug delivery can be divided into 3 groups: viscosity enhancing polymers which role is to increase the formulation viscosity by decreases in drainage time and increased bioavailability, Mucoadhesive Polymers which interacts with the Mucin layer by increase the contact time in eye and In-situ gelling polymers to get exposure to physiological condition in eye by undergoing sol-to-gel phase transition.

Colloidal Drug Delivery System
Colloidal Carriers are small particle system in which particle size in the range from 100 to 400 nm. As they are usually suspended in aqueous solution, they can easily administered as eye drops
Prodrugs

Figure 7 Approaches to ocular delivery
Ideal Characteristics of Ophthalmic Drug Delivery Systems:-
Must be sterile and isotonic.

Preservatives free.

Easy to use.

Well-organized Nasolacrimal Drainage.

Prolong contact time in eye after topical administration.

Superior Corneal Permeability.

Non-Irritant and having applicable Rheological Properties.

Increases the site specificity.

Reproducibility and Stability of Dosage Form.

Challenges to Ocular Drug Delivery:-
Formulation Considerations
1) Physiochemical Drug Properties
2) Buffer Capacity ; pH
3) Instillation Volume
4) Anti-Oxidants
5) Tonicity Adjustment
6) Surfactants
7) Preservatives
Anatomical and Physiological Considerations:-
1) Spillage of Drug by surplus
2) Dilution of Drug by Tears Turn over
3) Enzymatic Metabolism
4) Conjuctival Absorption
5) Nasolacrimal Drainage System
Pharmacokinetic Considerations:-
1) Transcellular Diffusion
2) Paracellular Diffusion
3) Charge of Intracellular Space

1.4 Disease Profile:-
1.4.1 Introduction of Disorders Related to Eye:-
There are various disease associated to eye like Muscular Disorders, Disorders related to Eye lid and Disorders related to globe of eye.

Muscular Disorders includes Nystagmus, Strabismus; Disorders of Eye lid like Blepharitis, Entropion, Conjunctivitis, Chalazion, Disorder related to globe of eye includes Glaucoma, Cataract, Corneal Abrasion, Uveitis, Diabetic Retinopathy and Dry Eye Disease.

DRY EYE DISEASE:-
It is a Multifactorial illness of Tears and Ocular Surface that results in symptoms of Visual Disturbance, Discomfort and Tear Film instability owed to damage to the ocular surface damage.

It is accompanied by Inflammation to ocular Surface and Increase in tear film osmolality.

It is also known as Keratoconjuctivitis Sicca.

Classification of Dry Eye Disease
nmTable SEQ Table * ARABIC 5 Classification of Dry Eye Disease
381000245110
Figure SEQ Figure * ARABIC 5 Difference between Dry Eye and Healthy Eye
Healthy Eye Dry Eye
It is Complex Mixture of proteins, Mucin and electrolytes. In this Condition Proteins and Growth factor concentrations decreased.

It contains Antimicrobial Proteins, Growth Factors, and suppressors of inflammation and Electrolytes for proper osmolality. Alteration in Cytokine balance which leads to Inflammation and Electrolytes are increases in this Condition.

Table SEQ Table * ARABIC 6 Difference between Dry ; Healthy Eye
1.4.2 Causes and Complications of Dry Eye:-
Dry eyes can also be caused by one’s environment, life style factors, certain physical abnormalities, and infections of the eye.

Risk Factors:-
Older Age Over 50 years of age group
Female Sex
Postmenopausal Status
Patient on Diuretics, Psychotropic, beta blockers medications are most susceptible to dry eye.

Patient exposed to heat and dust.

Environmental Stresses
Patient with Blepharitis.

Smoking and multivitamin use were associated with an increased risk of dry eye.

1.4.3 Sign and Symptoms:-
1) Discomfort
2) Dryness
3) Burning & Stinging
4) Foreign-Body Sensation
5) Gritty Feeling, Stickiness
6) Blurry Vision
7) Photophobia, Itching, Redness
8) Mucosal Discharge
9) Increased Frequency of Blinking
10) Diurnal fluctuation
1.4.4 Pathophysiology:-
It is accepted that the lachrymal glands, Ocular Surface, Lids and nerves that connect them are integral part to maintain tear production in eye.

Dysfunction of any of this results in unstable tear film that causes dry eye syndrome.

Dysfunction may be occur from natural ageing, Systemic Inflammatory diseases or surgeries that disrupts the nerves which are responsible to accelerate the tear production.

Diminished Tear Secretion produces inflammatory responses on ocular surfaces that involves the inflammatory mediators.

Inflammatory Mediators plays a key role in progression of Keratoconjuctivitis Sicca.
1.4.5 Diagnosis:-
For patients with mild irritation symptoms: a reduced tear breakup time (TBUT) may indicate an unstable tear film with normal aqueous tear production, and there may be minimal or no dye staining of the ocular surface.

For patients with moderate to severe symptoms: the diagnosis can be made by using one or more of the following tests:
• Tear break-up time (TBUT) test – to evaluate tear-film stability
• Ocular surface dye staining (Fluorescein/rose Bengal/Histamine green) test: to evaluate ocular surface disease (KCS)
• Schirmer test: to evaluate aqueous tear production these tests should be performed in this sequence because the Schirmer test can disrupt tear film stability and cause false-positive ocular-surface dye staining.

Treatment:-
The aims for treating dry eye disease include:
Reducing or alleviating signs and symptoms of dry eye
Maintaining and improving visual function
Reducing or preventing structural damage
Type of Therapy Treatment
Environmental Education and Environment modifications
Medication Topical Medication Artificial tear substitutes:- gels/ointments
Anti-inflammatory agents
Systemic Medication Omega 3 fatty acids
Tetracyclines
Surgical Punctal plugs
Permanent Punctal occlusion
Tarsorrhaphy
Other Contact Lenses
Eyelid Therapy
1.4.6 Current Scenario of Dry Eye Disease:-
The Below Study of Prevalence of Dry eye in U.S shows that In Asian and Hispanic Severe symptoms of DES are most seen compare to Diagnosed People.

White peoples are most clinically diagnosed from DES Compare to others.

Table SEQ Table * ARABIC 7 Prevalence of Dry Eye in U.S
Above age 80 men’s and women’s are more prone to DES compare to other age groups.

The Below Data Shows that Women’s are more sensitive to DES compare to Men.

4543425249809080-89
0080-89
3638550247904070-79
0070-79
2713990248856560-69
0060-69

Table SEQ Table * ARABIC 8 Prevalence by Age and Sex
The Incidence of Severe Dry Eye Disease over the age of 65 is around 75%.

Over Time Body produces less oil 65% less at age 65 to 70 than at age of 18.

1.4.7 Marketed Products for Treatment of Dry Eye Disease:-
Products Description Indication Year of Approval
0.03% ophthalmic solution of bimatoprost  0.005% benzalkonium chloride as preservative Used as Reduction of Elevated IOP. 2001
Travoprost 0.004% Ophthalmic Solution  0.015% benzalkonim chloride as preservative Reduction of IOP 2001
1% sterile aqueous topical ophthalmic solution of azithromycin 0.003% benzalkonim chloride as preservative Bacterial conjunctivitis 2007
Sterile topical ophthalmic gel containing 0.15% of ganciclovir 0.0075% benzalkonim chloride as preservative Herpetic keratitis 2009
0.45% ketorolac tromethamine solution Preservative Free ; contains CMC as Viscosity Enhancer. Used as NSAIDS After Cataract Surgery. 2009
0.05% of Difluprednate Emulsion formulated with boric acid, castor oil, glycerine, and Polysorbate 80, with 0.1% sorbic acid as preservative Steroid for treatment of inflammation and pain associated with Ocular Surgery. 2008
Table SEQ Table * ARABIC 9 Marketed Products
2.0 Aim and Objective:-,
Objective:-
Design, Development and characterization of ophthalmic emulsion of immunopressent drug Formulation of BCS Class- 4 using Microfluidizer as a technology.

Aim:-
Drug Substance Characterization
Innovator Product Characterization
Drug Excipient Compatibility Study
Selection of manufacturing process for development of Ophthalmic Emulsion
Preparation of ophthalmic Emulsion for a BCS class IV drug inline with the reference product.

Stability Study.

Rationale:-
To increase corneal penetration of drug.

To increase the site specificity of drug.

To formulate Preservative free formulation
Less nasocorneal drainage of system
Appropriate rheological properties
3.0 Drug and Excipients Profile:-
3.1 Introduction to Drug Substance
Table SEQ Table * ARABIC 10 Properties of Drug Substance
Drug Substance X
BCS Class 4
Category Topical Immunomodulator
Dosage Form Ophthalmic Emulsion
Appearance White Opaque to slight translucent homogeneous solution
Indication Keratoconjuctivitis Sicca
Bio-Availability 60%
Metabolism CYP450 3A
Half-life Approximately 7 hrs in children and 19 hrs in adults
Dose 0.05 and 0.10%
Onset of Action 12 hrs.

pH 6.5-8.0
Log P 4.20 to 4.30
Hygroscopicity Non-hygroscopic
Melting Point 148-151?c
Storage Conditions 15-25?c
Isomerism Chiral Centre Present
Photosensitivity Not sensitive to light
Mechanism of Action Tear Production is decreased when Lymphocytes, die and accumulate in tear glands. Drug X reverses this Condition by causing apoptosis of lymphocytes allowing for tear production.

3.2 Excipients Profile
The Decision of selection of specific excipients was further supported based on the results of the drug excipient compatibility study.

Table 11 List of Excipient used in formulation
Sr No. Name of Material Role
1 Drug A API
2 Castor Oil USP Solubilizing agent
3 Polysorbate 80 NF (Tween 80) Surfactant
4 Glycerin NF Tonicity Adjusting Agent
5 Polymer X Viscosity Enhancer/Stabilizer
6 0.1 N NaoH Neutralization
7 Milli Q Water Diluent
4.0 Literature Review:-
4.1 Literature review on Dose and Technology specific:
1) Przemyslaw Baranowski (2014) This article Describes Various Dosage form for Topical Ocular administration. It also prescribes In-vitro and In-vivo studies done for ophthalmic Dosage forms in order to determine whether the selected dosage forms meets the desired properties and patient’s Compliance.

2) Frederic Lallemand (2011) This article gives information about main steps which are involved in cationic nanoemulsion from formulation to evaluation in clinical trials.Major Hurdle of formulation was to selection of cationic agent which is having adequate safety profile which ensures efficient retention time of ocular surface. Toxicity and Pharmacokinetic Studies were performed to see whether cationic nanoemulsion are safe or not. Ongoing studies evaluating latanoprost emulsion in patients with ocular surface disease and glaucoma suggest that the beneficial effects on reducing ocular surface damage may also extend to this patient population.

3) Iskender Ince (2015) The Purpose of this article to develop a emulsion formulation
With appropriate physicochemical stability to enhance the bioavailability of pilocarpine.Various Evaluation parameters are studied and Results are shows that The developed formulation showed good physicochemical properties and a beneficial stability for six months. After microemulsion instillation into the rabbit eyes, the intraocular pressure was reduced significantly. The ocular irritation test used suggested that microemulsion formulation did not cause any significant allergies to the eye.

4.2 Literature Review on Drug and Excipient Specific:-
1) Anoop Kumar (2013) The Resolution of this article is to establish phase behaviour of different characterization methods available, type of Nanoemulsion, rheological properties and details of microstructure. Effect of Different components on final emulsion, recent trends on selection of excipients which contain oil phase, surfactant, and co-surfactant has been emphasised.

2) Philippe Piccerelle (2012) this article prescribes Novel ophthalmic delivery systems propose which use of many excipients to increase the viscosity or the bio adhesion of the ophthalmic emulsion. In this Articles newer Formulation of colloidal and gel dosage forms are made to study in vivo and invitro studies. The Results shows prolonged drug release and increase in drug bioavailability in eye, this type of drug delivery are promising approaches for ocular drug delivery. Relating different properties of pharmaceutical formulations appears to offer an unpretentious synergy in bioavailability and sustained release. Promising results are obtained with colloidal systems which present very comfortable conditions of use and prolonged action.

3) Mafi R (2014) the purpose is to develop an approach to predict the CFC for polymers. High molecular weight guar was hydrolysed to give a series of guar samples spanning a wide range of average molecular weights. The CFC values and critical viscosity concentrations were measured as functions guar properties, using electrophoresis, dynamic light scattering and rheology measurements. The higher the guar molecular weight, the lower was the CFC, the maximum concentration that can be tolerated in the eye drop formulation. The guar CFC values were approximately equal to the overlap concentrations where guar molecules start to overlap in solution. We propose that the CFC can be estimated for any water-soluble polymer using the polymer molecular weight and the readily available Mark-Houwink parameters, thus providing a design rule for ophthalmic emulsions.

4) Chang-wei Hsieh (2012) In this article response surface methodology used to predict the response of droplet of glabridin nanoemulsion. The results shows that The independent variables of homogenization pressure, Oil and Emulsifier Content and quadrics of 3 variables have big impact on globule size distribution. The Condition and concentrations of emulsifier tween 80 is 5.5%, Glycerol concentration 3.85% and homogenization.

5) Philippe Daull (2013) This article gives idea about the different approaches used to improve ocular bioavailability. It also includes roles of cationic agent in stabilization of safe cationic o/w nanoemulsion and benefits of cationic o/w nanoemulsion for protection and restoration of tear film and corneal epithelium.

4.3 Literature Review on Formulation specific:-
1) Yamaguchi M (2005) This article gives information that O/W emulsion is one of the approach to dissolve poorly water soluble drugs, A synthetic glucocorticoid Medicine Difluprednate is water insoluble compound containing Polysorbate 80 and Castor oil. Appearance of Emulsion was blue and translucent emulsion. The results shows the Mean of article size 105.0.After 6 months neither separation of 2 Phases and there is no change in particle size. Furthermore, when compared with Ophthalmic Suspension the Difluprednate ophthalmic emulsion shows 10 fold higher concentrations. This Emulsion is also useful for delivery of lipophilic drugs.

2) Noriaki Nagai (2014) This Article gives rationale of ophthalmic emulsion containing nanoparticles. In this article they provide a new method which is Bead mill method for preparation of Solid Nanoparticles using Indomethacin as active drug substance. The Particle size of this solid nano particle is around 65 to 85 nm and Dispersions of this nanoparticles are better tolerated by human and rat corneal epithelial cells than Conventional Eye Drops. Accumulations of drug in this solid Nanoparticles are more compare to solutions. The Furthermore Advantages are that solid nanoparticles doesn’t show any affect the antimicrobial activity against E.coli.The Corneal Absorption and Bioavailability is higher of this solid nanoparticles compare to conventional Nanoparticles. The Other most Benefit is this solid nanoparticles are having low cost. It seems that usage of this therapy expand the usage of this therapy.

3) R.R Nikhade (2015) The Present article give information about formulation, Evaluation and Validation of docosahexaenoic acid for ophthalmic delivery. By Preparing Conventional Dosage forms like capsule less amount of drug that reaches to inflammation site so more amount of dose is required, so to avoid this direct administration direct administration of drug into eye is done. The Formulation contains 4% DHA oil, 1.5% Tween 80, 0.5% Span 80.The Appearance of Emulsion was Milky White, the average particle size was 0.5µm and the viscosity was 40 cps as well as emulsion was stable at 25?c and 40?c for more than 6 months. Moreover the formulated emulsion was validated for the physical properties which are evaluated. The analysis of DHA in the emulsion was done by gas chromatography.

4) Vivek P Chavda (2016) This article focuses on Nanoemulsion as a drug delivery vehicle. Nanoemulsion provide benefits like protect labile drug, control drug release, Increase drug solubility and Bioavailability and diminishes patient variability. Lipophilic Mixture having particle size less than 100 nm.Nanoformulation provide various options to develop Nano formulation for different drug and administration routes. This strategy also used to evaluate efficacy of toxic drugs.

5) G.Aiswarya (2015) This article is provide information about topical gel loaded nanoemulsion containing flurbiprofen using volatile oil. The selection of oil phase, surfactant and cosurfactant was carried out by screening method with additional of pseudo-ternary phase study.32 factorial designs were applied with using response surface methodology. Formulation was prepared by using spontaneous emulsification method. Various statistical data were investigated. From Data it was found that the selected formulation shows optimum in-vivo data and then compound finally compare to marketed formulations.

4.4 Literature Review on Patent:-
1) Patent No: – US 2008/0181867 A1
Published Date:-Jul.31, 2008.

Gregory Lambert The Present Development communicates to newer processes for preparations. Number of processes are provided which includes pre-concentrate of oil in water emulsion and diluting the pre-concentrate which is obtained from o/w emulsion. It also provides different pharmaceutical compositions containing o/w emulsion which is prepared by innovative process. Such type of inventory process have advantage like low energy output than required and various methods use for treatment of eye disease. In Present invention the step of emulsification done by using high pressure homogenizers or sonicating homogenizers. Emulsification step requires energy output lower which is 4 to 50 times lower than require.

2) Patent No:- WO2006050837 A2
Published Date:-May 18, 2006.

Betty Philips This invention refers to ophthalmic emulsions which is containing immunosuppressive agent as an active ingredient in oil which is suitable for eye conditions. According to this invention dry eye disease is ocular dryness and tear deficiency. It is not limited to condition of excessive evaporation of tears or any conditions related to defective tear film.or Conjuctival dryness which can be done due to insufficient production of tears. And conditions results are corneal keratitis and corneal epithelial erosion.

5.0 Materials and Methods:-
5.1 List of Equipments:-
List of equipment’s used or proposed to be used in the current research project are given in the table below:
Table:12 List of the Equipments used in the formulation & development
Sr. no Equipments Model and manufacturer
1 Digital Stirrer Remi equipment India
2 IKA Homogenizer IKA Ultra-Turrax Homogenizer T-18
3 High Pressure Homogenizer Panda
4 Microfluidizer M-110 P,Microfluidics Corporation
5 Zetasizer Malvern
6 HPLC Agilent 1100
7 PH Meter Orion star A211, thermo fisher
8 Brookfield Viscometer Malvern
Microfluidizer Process:
541020031750OUTLETT
00OUTLETT

5191125725805COOLING JACKET
00COOLING JACKET
2019300192405Intensifier Pump
00Intensifier Pump
3524250821055Interaction Chamber
00Interaction Chamber
43668952176780Pressure up to 276 Mpa (40000 psi)
00Pressure up to 276 Mpa (40000 psi)
-5207001129030Product
00Product
126365309880Inlet Reservoir
00Inlet Reservoir

Advantages of Microfluidizer Technology:-
It produce smaller particles when compares with other technology.

It has much tighter particle size distribution.

It has Fixed Geometry.

It has greater Repeatability.

Microfluidizer generates high shear compare to other technology like high pressure homogenizer.

Microfluidizer has having higher Emulsification Efficiency Compare to High Pressure Homogenizer.

6.0 Methodology:-
6.1 Preparation of Drug Phase:-
Take Quantity of Castor Oil and Polysorbate 80 in s.s vessel, heat the mixture with stirring till 55-60?c.

When Temperature reaches to 55-60?c, Add Drug Substance to it under stirring. Continue Stirring till Complete solublization of drug substance. Maintain temperature 55-60?c during stirring.

6.2 Preparation of Polymer Phase:-
Take 10 gm of Polymer X and add to vortex of 1300 gm of Milli Q water under stirring at 2000 rpm stir for 30 to 40 mins.

Take 6880 gm of Milli Q water stir to form vortex,Add step 1 Premix to it,If required ,Rinse Premix Vessel with Milli Q water and stir the solution for 2 hr at 2000 rpm.

Sterilization of Polymer Phase:-
After Stirring of Polymer Phase for 2 hr, sterilization is done for 121?c for 20 mins.
6.3 Preparation of Primary Emulsion:-
Take Drug Phase and heat it to about 55-60?c.

Take Milli Q Water and add Glycerin to it .Homogenise this Phase with heating up to 45-50?c.

When Temperature reaches to 45-50?c of Glycerin phase add Drug Phase into it, Rinse the drug phase vessel with Milli Q water and added to above phase.

Homogenize this Phase at 9600 rpm for 15 mins maintain temperature during homogenization to 45-50?c.

After Completion of Homogenization, Pass this Coarse Emulsion through Microfluidizer at 20000 Psi. Maintain Coarse Emulsion Temperature 40-45?c and Outlet temperature 25-30?c.

After Passing of all solution pass Milli Q Water into Microfluidizer for Stirring Purpose.

Stirr Primary Emulsion at 250-350 rpm and add Milli Q water to it and Stirr for 10 mins at 250 rpm.

6.4 Preparation of 0.1 N NaoH Solution:-
Dissolve 2 g of NaoH Pellets into 500 g of Milli Q water into it and stirr for 20 mins.

6.5 Neutralization Part:-
Take Primary Emulsion and add require quantity of Polymer Phase into it and stirr for 20 mins at 350-400 rpm.

After Completion of mixing add 40 g of 0.1 N NaoH into it and stirr for 1 hr at 1200-1300 rpm.

After Stirring for 1 hr add 20 g of 0.1 N NaoH into it and stirr for 2 hr at 1200-1300 rpm.7F
FFFJFFJFJFFJS20030165545A
7 Experimental Work:-
7.1 Preformulation Study of Drug:-
7.1.1 Drug Solubility Study
Solubility study was performed in different medium at 25°C using shake flask method to determine the solubility of drug in different pH. For details of the solubility of the drug are mentioned in Table 13.

Media Solubility (mg/ml) Media Solubility (mg/ml)
Ethyl Acetate 750 pH 1.2 0.02
Acetone 100 pH 3.0 0.03
Ethanol 150 pH 4.6 0.05
Water 0.01 pH 6.8 0.03
– – pH 8.0 0.04
Table:-13 Drug Substance Solubility in Various Media
Result and Discussion: – From solubility data in various media, it was found that Drug A is poorly soluble in water.

7.1.2 Physicochemical properties of Drug A:-
7.1.2 Innovator Product Characterization
Innovator product characterization was carried out by physical and chemical evaluation of innovator product. It include description, pH, assay, viscosity and Globule size distribution. The information is summarised in Table no 14.

Table:- 14 Reference Product Characterization
Tests Observations
pH 6.4 to 7.8
Viscosity 50 to 55 cps
Globule Size Distribution d(10)=99 to 105 nm
d(50)=440 to 465nm
d(90)=1840 to 1855 nm
Avg. Diameter= 342 to 360 nm
Span Value (D90-D10/D50) 3.20 to 3.39
PDI 0.28 to 0.35
Osmolality (mOsml/L) 302-310
Surface Tension (N/m) 0.038 to 0.045
Zeta Potential (mv) -30.67 to -37.12
Drop Weight Wt.(mg)at 45?C=23.65 to 25.23 mg,wt.(mg)at 90?C=22.12 to 23.54 mg
7.1.3 Drug Excipient Compatibility Study:-
To study the compatibility of the excipients with Drug X, drug-excipient compatibility studies were undertaken for Drug X with different excipients. During the course of the study, any change in degradation product profile was monitored. The procedure employed for drug-excipient compatibility studies is briefly summarized below.

Brief Procedure: Drug and individual excipients were mixed in a predetermined ratio. The triturated blend was transferred to glass vials capped with rubber closure sealed with aluminium seal (closed condition). The samples were then kept under accelerated condition of 40°C±2?C/ 25% RH±5?C up to 1 month, Intermediate Condition 30°C±2?C/ 65% RH±5?C up to 1 month and Long term Condition 25°C±2?C/40% RH±5?C up to 1 month. The results of drug-excipients compatibility studies are shown in Table 15.

Table 15 Drug Excipient Compatibility Study
Drug Substance/Name of Excipient 102552525971500Drug: Excipient Ratio 68453125273000 Quantity (mg)
DS Excipient Mili Q (ml)
Drug X + Milli Q water NA 5.00 NA 20.00
Drug+ Polysorbate 80+Milli Q water 1:30 5.00 150.0 20.00
Drug+ Castor oil+Milli Q water 1:25 5.00 125.0 20.00
Drug +Glycerin+ Milli Q water 1:40 5.00 200.0 20.00
Drug+Polymer+ Milli Q water 1:2 5.00 100.0 20.00
Drug+Sodium Hydroxide Solution+ Milli Q water 1:80.07 5.00 400.35 20.00
Castor oil+Milli Q water NA NA 125.0 20.00
Polysorbate 80+Milli Q water NA NA 150.0 20.00
Drug +Glycerin+ Milli Q water NA NA 200.0 20.00
Polymer+ Milli Q water NA NA 100.0 20.00
Sodium Hydroxide Solution+ Milli Q water NA NA 400.35 20.00
Results:-
Storage Condition Study Duration Testing Frequency Description pH Related Substances
40?c±2?c/25±5%RH 1 month Initial Milky white Emulsion 7.32 0.42%
15 days Milky white Emulsion 7.20 0.45%
1 month Milky white Emulsion 7.11 0.53%
30?c±2?c/65±5%RH 1 month 1 month Milky white Emulsion 7.24 0.45%
25?c±2?c/40±5%RH 1 month 1 month Milky white Emulsion 7.16 0.47%
Discussion:-
Drug-Excipient compatibility study did not show significantly higher degradation products at various storage conditions when compared with the drug substance. Hence, it can be inferred that the above investigated excipients can be used for the purpose of formulation.

7.5.1 Forced Degradation Studies:-
Forced degradation studies are carried out for the following reasons like to develop and validate a stability indicating method, to determine degradation pathways and impurities during development phase, to understand the drug molecule chemistry and by generating degradation profile of drug product and to solve stability-related problems (e.g. Mass balance)
The Conditions are mentioned in Table 16.

Table 16 Stress and Force Degradation Study
Stress Condition Highest Known Highest Unknown Total Impurities
Oxidation
(Samples treated with 2 ml 1% hydrogen peroxide at 40?c.) BQL 0.05 0.05
Acidic Hydrolysis
(Samples treated with 2 ml, 5 M HCl at room temperature for 9 days.)
BQL 0.05 0.05
Base Hydrolysis
(Samples treated with 2 ml, 10 M NaoH at R.T for 9 days. BQL 0.05 0.05
Thermal Stress Condition
(60?c for 7 days.) BQL BQL BQL
Humidity Stress Condition
(40°C/75% RH For 7 Days) BQL BQL BQL
Discussion: – Drug Substance had no degradation in assay & organic impurities when exposed to thermal and humidity stress condition. It was sensitive to acidic, alkaline and oxidation condition.

7.1.4 Container Closure Study:-
Container Closure study was done to study assay and related substance of ophthalmic emulsion at 24 hr, 48 hr and 72 hr. and study was carried out the component, filter and saint Gobain Compatibility.

Table 17 Container Closure study for measurement of Assay & Related Substance
Pack:- 5.5 ml in 11 ml HDPE Bottle
Tests Time Component Compatibility Filter Compatibility Tubing study
Assay
24 hr 104.4 103.5 102.8
48 hr 104.0 102.4 101.9
72 hr 103.7 102.2 100.8

Pack:- 5.5 ml in 11 ml HDPE Bottle
Tests Time Component Compatibility Filter Compatibility Tubing study
Related Substance 24 hr 0.86 0.82 0.76
48 hr 0.88 0.86 0.79
72 hr 0.89 0.87 0.83
Discussion: – From the results, it was concluded there was no any significant change in Assay and Related Substance of ophthalmic emulsion with increase in time.

7.2 Formulation Strategy:-
7.2.1 Design of Formulation:-
34289918224500
97155036131500157162523749000619125660400017659351714500
285496093345Aqueous Phase
00Aqueous Phase
56769017526000
-273050121920Drug+Oil Phase
00Drug+Oil Phase

225425053975Emulsifying Agent
00Emulsifying Agent

Figure SEQ Figure * ARABIC 8 Formulation Design
7.2.2 Critical Process Parameters:-
Summary of medium to high risk critical process parameters involved in process of preparing ophthalmic emulsion are listed below Table. Along with the list of tests /action required to address critical level. These CPP were obtained from the previous experience and trial batches performed.
Table no.18 Critical process parameters for preparation of ophthalmic emulsion
Unit Operation Parameters Proposed Range of Data
Polymer Phase Stirring Time 2 to 3 hrs.

Stirring Speed 600-800 USM
Drug Phase Temp. of Castor oil & Glycerin 55-60?C
Bulk Sterilization of Polymer Phase Sterilization Temp. 121?C
Sterilization Time 30 mins.

Temperature of Cooling water 20-25?C
Addition of Primary Emulsion to Polymer Phase Stirring time 20-25 min.

Stirring Speed 450-550 USM
Filtration of 0.1 N NaoH Solution and Neutralization of Bulk Emulsion Stirring time Part-1- 1 hr and Part-2- 2 hr.

Stirring Speed 1400-1600 USM
Sequence of pH adjustment Part-1- 70% and Part-2- 30%.

Final Stirring Stirring time 1400-1600 USM
Stirring Speed 3 to 4 hrs.

7.3 Preliminary Trials Using QbD approach:-
7.3.1 Trial Batch containing Drug A, Polymer X using IKA homogenizer.
Table 19 Formulation of batch 1:-
Sr No. Ingredients %w/v
1 Drug A 0.10%
2 Castor Oil USP 1.10%
3 Polysorbate 80 USP 1.30%
4 Glycerol USP 7.50%
5 W.F.I Q.S…

6 Polymer Phase 2.5%
7 0.1 N NaoH solution 4.2%
Table 20 Conditions of IKA homogenizer:-
Variables Conditions
Homogenization speed 8200 rpm
Homogenization Time 20 min
Table 21 Observations of batch 1:-
Tests Observations
Description White Opaque to slight translucent homogeneous solution
pH 6.23
Viscosity 28.40 cps
Osmolality 273 mOsml/L
Results and Discussion: – Unstable System with low pH and viscosity data was obtained.

7.3.2 Trial Batch of Drug phase Stability at different time.

Table 22 Formulation of batch 2:-
Sr No. Ingredients %w/v
1 Drug X 0.10%
2 Castor Oil USP 1.10%
3 Polysorbate 80 1.30%
Table 23:- Observation of batch 2:-
Tests Drug Phase Temperature 50-55?C
30 Mins. 60 Mins. 90 Mins. 120 Mins.

Related Substance (%) 0.45% 0.45% 0.46% 0.46%
Result and Discussion: – It was concluded that there is no trend of increase in impurity was found up to 2 hrs by keeping drug phase temperature 50-55?C.

7.3.3 Effect of time of mixing on polymer phase property.

Table 24 Formulation of batch 3:-
Step:-1
Sr No. Ingredients %w/v
1 Polymer X 0.10%
2 Milli Q water 14.5%
Step:-2
Sr No. Ingredients %w/v
1 Step (1) formulation –
2 Milli Q water 60.40%
Table 25 Evaluation of batch 3:-
Variables Viscosity(cps)
Polymer Premix with 15 min. mixing time 1720
Polymer Premix with 30 min. mixing time 1130
Polymer Premix with 45 min. mixing time 836.5
Polymer Premix with 60 min. mixing time 802.3
Note:- Viscosity of polymer premix was measured at 30 rpm using spindle S18.

Results and Discussion:- From the evaluation parameters we concluded that as premix time of polymer phase increased, the viscosity of emulsion simultaneously decreased.

7.3.4 Effect of Pressure on emulsion using High pressure homogenizer.

Table 26 Formulation of batch 4:-
Sr No. Ingredients %w/v
1 Drug A 0.10%
2 Castor Oil USP 1.10%
3 Polysorbate 80 USP 1.30%
4 Glycerol USP 2.50%
5 W.F.I Q.S…

6 Polymer Phase X 2.5%
7 0.1 N NaOH 4.2%
Table 27 Evaluation of emulsion using HPH:-
Particle Size Distribution
Conditions
(bar) D(10)
(nm) D(50)
(nm) D(90)
(nm) Avg.Diameter
(nm) PDI Span
500 166.6 206.7 420.7 208.8 0.308 1.62
1000 162.9 212.3 625.5 190.3 0.293 2.82
1500
162.9 176.6 400.7 177.7 0.245 1.50
Result and Discussion:- As pressure increase, Globule size of emulsion decreases. But the target globule size is not achieved so subsequent trials are taken by using Microfluidizer.
7.3.5 Emulsion preparation by changing the Process Variables using Microfluidizer.

Formulation of batch 5:-
The formulation for batch 5 is same as in batch 4, only processing conditions were varied for batch 5.The process variables are mentioned in table 28.

Table 28 Process Variables for batch 5:-
Variables Conditions
Drug:Glycerol Ratio 1:7
Homogenization Speed 8200 rpm
Homogenization Time 10 min.

Microfluidization Pressure 1724 bar
Coarse emulsion temperature 30?C
Outlet temperature 25-30?C
Number of cycles 1
Table 29 Evaluation by varying process variables:-
Tests Observations
pH 7.43
Viscosity 52.9 cps
Globule Size Distribution D(10)=142.1 nm,D(50)=548.7 nm,D(90)=2077.7 nm
Avg.=449.7 nm
PDI 0.345
Span 3.60
Related Substance 0.95%
Assay 100.6%
Surface Tension (N/m) 0.05199
Osmolality (mosml/L) 278
Zeta Potential (mv) -29.42
Drop Weight Wt.(mg)at 45?=28.23;wt.(mg)at 90?=25.36
Result and Discussion:- By preparing emulsion using Microfluidization technology and increase in Microfluidization pressure from 1500 bar to 1724 bar the globule size of emulsion was found to be on higher side and pH found to be 7.43 which also considered as high.

7.3.6 To Prepare Final Ophthalmic Emulsion by varying the Number of Cycles.

Formulation of batch 6:-
The formulation for batch 6 is same as in batch 4, only processing conditions were varied for batch 6.The process variables are mentioned in table 30.

Table 30 Operating Conditions:-
Variables Conditions
Drug:Glycerol Ratio 1:7
Homogenization Speed 8200 rpm
Homogenization Time 10 min.

Microfluidization Pressure 25000 psi.

No. of cycles 2
Coarse emulsion temperature 30?C
Outlet Temperature 25-30?c
Table 31 Evaluation of ophthalmic emulsion by changing number of cycles:-
Tests Observations
pH 7.35
Viscosity 50.3 cps
Globule Size Distribution D(10)=134.1 nm,D(50)=536.7 nm,D(90)=1987.8 nm
Avg.=449.7 nm
PDI 0.342
Span 3.57
Related Substance 0.88%
Assay 100.2%
Surface Tension (N/m) 0.04969
Osmolality (mOsml/L) 287 mOsml/L
Zeta Potential (mv) -30.23
Drop Weight Wt.(mg)at 45?=30.23;wt.(mg)at 90?=27.67
Result and Discussion:-From evaluation parameters we concluded that as number of cycles increased globule size decreased but there was no significant change seen in pH, viscosity and zeta potential of emulsion.

7.3.7. Effect of changing homogenization speed and time on emulsion preparation.

Formulation of batch 7:-
The formulation for batch 7 is same as in batch 4, only processing conditions were varied for batch 7.The process variables are mentioned in table 32.

Table 32 Operating conditions:-
Variables Conditions
Drug:Glycerol Ratio 1:7
Homogenization Speed 10500 rpm
Homogenization Time 15 min.

Microfluidization Pressure 25000 psi.

Coarse emulsion temperature 50?c
Outlet temperature 25-30?c
No. of cycles 1
Table 33 Evaluation by changing homogenization speed and time:-
Tests Observations
pH 7.37
Viscosity 48.8 cps
Globule Size Distribution D(10)=119.9 nm,D(50)=522.8 nm,D(90)=1840.8 nm
Avg.=419.9 nm
PDI 0.340
Span 3.50
Related Substance 0.85%
Assay 100.5%
Surface Tension (N/m) 0.05341
Osmolality (mOsml/L) 293
Zeta Potential (mv) -31.65
Drop Weight wt.(mg)at 45?=30.90;wt.(mg)at 90?=26.66
Result and Discussion:- As increased in homogenization speed from 7600 to 10500 rpm and increased in homogenization time from 10 min to 15 min, there was significant decrease in diameter of globule size and viscosity of emulsion, However Osmolality value and pH was not within the normal range for eye.

7.3.8 Effect of temperature of polymer phase which kept at room temperature when added in drug phase.

Formulation of batch 8:-
The formulation for batch 8 is same as in batch 4, only processing conditions were varied for batch 8.The process variables are mentioned in table 34.

Table 34 Operating conditions:-
Variables Conditions
Drug:Glycerol Ratio 1:7
Homogenization Speed 10500 rpm
Homogenization Time 15 min.

Microfluidization Pressure 26000 psi.

Polymer phase temperature 25?
Coarse emulsion temperature 40?c
Outlet temperature 60-70?c
No. of cycles 1
Table 35 Evaluation of ophthalmic emulsion by keeping polymer phase at room temperature.

Tests Observations
pH 7.33
Viscosity 49.9 cps
Globule Size Distribution D(10)=127.6 nm,D(50)=516.8 nm,D(90)=2023.8 nm
Avg.=426.7 nm
PDI 0.319
Span 3.64
Related Substance 0.63%
Assay 100.2%
Surface Tension (N/m) 0.05142
Osmolality (mOsml/L) 297
Zeta Potential (mv) -29.67
Drop Weight wt.(mg)at 45?=30.10;wt.(mg)at 90?=26.87
Result and Discussion:-By kept polymer phase at room temperature and coarse emulsion decreased from 50?C to 40?C,It observed that globule size distribution of emulsion was on higher side when compared with previous batch 7.

7.3.9 Effect of temperature of polymer phase which kept at 70?C when added in drug phase.

Formulation of batch 9:-
The formulation for batch 9 is same as in batch 4, only processing conditions were varied for batch 9.The process variables are mentioned in table 36.

Table 36 Operating conditions:-
Variables Conditions
Drug:Glycerol Ratio 1:7
Homogenization Speed 10500 rpm
Homogenization Time 15 min.

Microfluidization Pressure 26000 psi.

Polymer phase temperature 70?
Coarse emulsion temperature 40?c
Outlet temperature 60-70?c
No. of cycles 1
Table 37 Evaluation of ophthalmic emulsion by keeping polymer phase at room temperature.

Tests Observations
pH 7.28
Viscosity 48.8 cps
Globule Size Distribution D(10)=117.5 nm,D(50)=475.0 nm,D(90)=1922.7 nm
Avg.=399.1 nm
PDI 0.329
Span 3.76
Related Substance 0.68%
Assay 99.6%
Surface Tension (N/m) 0.04987
Osmolality (mOsml/L) 299
Zeta Potential (mv) -28.02
Drop Weight wt.(mg)at 45?=29.97wt.(mg)at 90?=26.90
Result and Discussion:-By kept polymer phase at 70?C and coarse emulsion decreased from 50?C to 40?C,It observed that globule size distribution of emulsion was about D(10),D(50),D(90) obtained 117.5,475.0,1922.7 nm which was decreased compared to previous batch 8 in which globule size of emulsion was found to be 127.6,516.8,2023.8 nm. There was no significant change in pH, viscosity data of batch 8 and 9.

7.3.10 Effect of Changing the Drug: glycerol Ratio, Homogenization Time and Speed using microfluidizer.

Table 38 Formulation of batch 10:-
Sr No. Ingredients %w/v
1 Drug A 0.10%
2 Castor Oil USP 1.10%
3 Polysorbate 80 USP 1.30%
4 Glycerol USP 3.50%
5 W.F.I Q.S…

6 Polymer Phase 2.5%
7 0.1 N NaoH 4.2%
Table 39 Operating Conditions:-
Variables Conditions
Drug:Glycerol ratio 1:4
Homogenization speed 8200
Homogenization time 10 min.

Microfluidization pressure 25000 psi.

Coarse emulsion temperature 30?C
Outlet temperature 25-30?C
No. of cycles 1
Table 40 Evaluation by changing Drug: glycerol ratio, Homogenization speed and
time:-
Tests Observations
pH 7.30
Viscosity 48.2 cps
Globule Size Distribution D(10)=114.3 nm,D(50)=433.7 nm,D(90)=1637.8 nm
Avg.=381.6 nm
PDI 0.345
Span 3.68
Related Substance 0.83%
Assay 101.2
Surface Tension (N/m) 0.05235
Osmolality (mOsml/L) 298
Zeta Potential (mv) -33.02
Drop Weight Wt.(mg)at 45?=30.93;wt.(mg)at 90?=26.89
Result and Discussion:-By changing in Drug: glycerol ratio from 1:7 to 1:4 which was kept in batch 7 and decreased in homogenization speed and time and maintaing microfluidization pressure same as that was in previous batch there was slightly decreased in globule size, pH, viscosity.

7.3.11 Trial Batch Keeping Coarse Emulsion Temperature 60?C.
Formulation batch 11:- The formulation for batch 11 is same as in batch 10, only processing conditions were varied for batch 11.The process variables are mentioned in table 41.

Table 41 Operating Conditions:-
Variables Conditions
Drug:Glycerol ratio 1:4
Homogenization speed 10200
Homogenization time 20 min.

Microfluidization pressure 25000 psi.

Coarse emulsion temperature 60?C
Outlet temperature 30-35?C
No. of cycles 1
Table 42 Evaluation by changing in Coarse emulsion temperature, homogenization speed and time:-
Tests Observations
pH 7.35
Viscosity 54.6 cps
Globule Size Distribution D(10)=107.2 nm,D(50)=463.4 nm,D(90)=1870.8 nm
Avg.=381.6 nm
PDI 0.349
Span 3.84
Related Substance 0.89%
Assay 99.1%
Surface Tension (N/m) 0.05235
Osmolality (mOsml/L) 298
Zeta Potential (mv) -29.67
Drop Weight Wt.(mg)at 45?=25.87;wt.(mg)at 90?=23.31
Result and Discussion:- As coarse temperature of emulsion increased 60?C from 30?C and subsequently increased homogenization speed 10200 from 8200 and time 20 mins instead of 10 mins which kept in previous batch 9, it was found that there was decrease in globule size of emulsion and viscosity increased, there was no change in osmolality.

7.3.12 Trial by varying in Microfluidization pressure and coarse emulsion temperature.

Table 43 Formulation of batch 12:-
Sr No. Ingredients %w/v
1 Drug A 0.10%
2 Castor Oil USP 1.10%
3 Polysorbate 80 USP 1.30%
4 Glycerol USP 2.50%
5 W.F.I Q.S…

6 Polymer Phase 2.5%
7 0.1 N NaoH 3.8%
Table 44 Operating Conditions:-
Variables Conditions
Drug:Glycerol ratio 1:4
Homogenization speed 10200
Homogenization time 20 min.

Microfluidization pressure 21000 psi.

Coarse emulsion temperature 40-45?C
Inlet temperature 25-33?C
No. of cycles 1
Tests Observations
pH 7.34
Viscosity 60.6 cps
Globule Size Distribution D(10)=105.3 nm,D(50)=469.7 nm,D(90)=1978.3 nm
Avg.=389.1 nm
PDI 0.348
Span 3.90
Related Substance 0.82%
Assay 101.6%
Surface Tension 0.05123
Osmolality 299 mOsml/L
Zeta Potential -30.90
Drop Weight Wt.(mg)at 45?=29.93;wt.(mg)at 90?=25.44
Table 45 Evaluation of varying in Microfluidization pressure and coarse emulsion temperature.

Result and Discussion: – As Microfluidization pressure and coarse emulsion temperature decreased which was kept 25000 PSI and 60?C respectively in previous batch 9, it was found that the globule size of emulsion decreased but there was no decrease in D(90) which was obtained 1978.3 nm which was 1870.8 nm in previous batch 9 and viscosity of emulsion obtained 60.6 cps of this batch which was higher compared to previous trial where viscosity found around 45 to 55 cps.

7.3.13 Trial batch by varying in holding time of emulsion.

Formulation of batch 13:- The formulation for batch 13 was same as in batch 10, only processing conditions were varied for batch 13. The process variables are mentioned in table 46.

Table 46 Evaluation of different holding time on pH and viscosity:-
Holding time after addition of drug in polymer phase pH Viscosity
(cps)
0 min hold 6.0 39.5
30 min hold 6.6 36.2
60 min hold 7.0 52.1
90 min hold 7.10 66.8
120 min hold 7.27 47.5
Results and Discussion:-After addition of drug phase in polymer phase and then varying in holding time at 0,30,60,90,120 min,it concluded that as holding time increased, there was no significant change observed in pH,Viscosity.

7.3.14 Trial batch by changing in coarse emulsion temperature and keeping hold time of emulsion 1 hr.

Formulation of batch 14:- The formulation for batch 14 was same as in batch 10, only processing conditions were varied for batch 14. The process variables are mentioned in table 45.

Variables Conditions
Drug:Glycerol Ratio 1:4
Homogenization Speed 10200
Homogenization Time 20 min.

Microfluidization Pressure 21000 psi.

Coarse Emulsion Temperature 60-65?C
Table 47 Operating Conditions:-
Table 48:-Evaluation by changing coarse emulsion temperature and keeping hold time 1 hour of emulsion.
Tests Observations
pH 7.23
Viscosity 48.09 cps
Globule Size Distribution D(10)=102.3 nm,D(50)=464.7 nm,D(90)=1976.1 nm
Avg.=389.1 nm
PDI 0.320
Span 3.86
Related Substance 0.74%
Assay 100.3%
Surface Tension (N/m) 0.05021
Osmolality (mOsml/L) 304 mOsml/L
Zeta Potential (mv) -30.23
Drop Weight Wt.(mg)at 45?=24.93;wt.(mg)at 90?=25.32
Results and Discussion:- As coarse emulsion temperature increased (60-65?C) which was kept 40-45?C in previous batch 13 and after adding primary emulsion into polymer phase kept holding time 1 hour, it was found that globule size of emulsion decreased which is D(10),D(50),D(90)which is 102.3 nm,464.7 nm,1976.1 nm, simultaneously pH and viscosity found to be 7.23 and 48.09,osmolality was found to be 304 mosml/L.PDI and Zeta potential is found to be 0.320 and -30.23 mv.In innovator batch globule size D(10),D(50),D(90) was about 99 to 105 nm,440 to 465 nm,1840 to 1855 nm respectively, pH and viscosity was about 6.4 to 7.8 and 50 to 55 cps.osmolality and PDI was 302 mosml/L and -30.67 mv. So when innovator data compared with batch 14 it concluded that data obtained in this batch were similar to innovator data. So, batch 14 considered as optimized batch.

7.6 Stability Study of Optimized Formulation:-
Table 49 Different Accelerated stability condition (40?C±2?C/25±5%RH) of Optimized formulation
Tests Initial 15 days 1 M 2 M 3 M 6 M
Storage Condition :- 40?C±2?C/25±5%RH ,Upright With Label
Pack Description:- 5.5 ml in 11 ml LDPE Bottle
pH 7.21 7.19 7.16 7.13 7.12 7.12
Weight per ML 1.0037 1.0036 1.0036 1.0037 1.0035 1.0033
Viscosity 54.67 cps 54.0 cps 53.1 cps 53.0 cps 52.5 cps 51.8 cps
Osmolality 303 mosm/l 283 mosm/l 278 mosm/l 280 mosm/l 279 mosm/l 277 mosm/l
Assay (%) 96.8 % 96.0 % 95.7 % 95.3 % 94.8 % 94.4 %
Globule Size distribution
(nm) D(10)=102.4
D(50)=464.8
D(90)=1976.6 D(10)=101.7
D(50)=461.5
D(90)=1972.5 D(10)=100.9
D(50)=460.8
D(90)=1970.6 D(10)=100.2
D(50)=460.1
D(90)=1969.0 D(10)=100.0
D(50)=459.8
D(90)=1967.3 D(10)=97.2.

D(50)=455.6
D(90)=1960.6
PDI 0.316 0.313 0.310 0.309 0.309 0.308
Span 3.86 3.85 3.83 3.83 3.82 3.80
Zeta Potential -31.12 mv -31.05 mv -30.76 mv -30.50 mv -29.64 mv -27.21 mv
Related Substance 0.42% 0.45% 0.49% 0.54% 0.55% 0.60%
Tests Initial 15 days 1 M 2 M 3 M 6 M
Storage Condition :- 40?C±2?C/25±5%RH,Upright Without Label
Pack Description:- 5.5 ml in 11 ml LDPE Bottle
pH 7.21 7.09 7.08 7.06 7.03 7.01
Weight per ML 1.0037 1.0034 1.0034 1.0032 1.0030 1.0029
Viscosity 54.67 cps 53.40 cps 53.1 cps 52.9 cps 52.4 cps 51.2 cps
Osmolality 303 mosm/l 275 mosm/l 272 mosm/l 270 mosm/l 269 mosm/l 267 mosm/l
Assay (%) 93.8 % 93.0 % 92.7 % 92.3 % 91.8 % 91.4 %
Globule Size distribution
(nm) D(10)=102.4
D(50)=465.8
D(90)=1977.6 D(10)=102.0
D(50)=465.2
D(90)=1976.6 D(10)=101.3
D(50)=464.8
D(90)=1976.1 D(10)=101.0
D(50)=464.5
D(90)=1975.3 D(10)=100.8
D(50)=464.0
D(90)=1973.1 D(10)=100.6
D(50)=463.7
D(90)=1972.6
PDI 0.316 0.341 0.343 0.345 0.349 0.354
Span 3.86 3.85 3.84 3.78 3.76 3.73
Zeta Potential -31.12 mv -30.05 mv -29.76 mv ±28.50 mv -27.64 mv -27.26 mv
Related Substance 0.42% 0.49% 0.53% 0.57% 0.65% 0.67%
Tests Initial 15 days 1 M 2 M 3 M 6 M
Storage Condition :- 40?C±2?C/25±5%RH,Inverted Without Label
Pack Description:- 5.5 ml in 11 ml LDPE Bottle
pH 7.21 7.03 7.00 6.96 6.92 6.90
Weight per ML 1.0037 1.0030 1.0027 1.0026 1.0023 1.0020
Viscosity 54.67 53.0 52.1 51.9 51.3 50.0
osmolality 303 272 268 263 260 257
Assay (%) 93.3 92.8 92.2 92.0 91.2 90.9
Globule Size distribution D(10)=102.4
D(50)=465.8
D(90)=1977.6 D(10)=101.0
D(50)=465.0
D(90)=1975.6 D(10)=100.9
D(50)=463.8
D(90)=1975.1 D(10)=100.8
D(50)=463.5
D(90)=1975.0 D(10)=100.7
D(50)=463.0
D(90)=1973.1 D(10)=100.6
D(50)=461.7
D(90)=1970.6
PDI 0.316 0.355 0.358 0.361 0.364 0.369
Span 3.86 3.83 3.80 3.78 3.76 3.73
Zeta Potential -31.12 -29.05 -29.02 -28.34 -27.12 -26.49
Related Substance 0.42% 0.58% 0.63% 0.66% 0.68% 0.69%
Tests Initial 15 days 1 M 2 M 3 M 6 M
Storage Condition :- 40?C±2?C/25±5%RH,Inverted with Label
Pack Description:- 5.5 ml in 11 ml LDPE Bottle
pH 7.21 7.15 7.13 7.10 7.07 7.03
Weight per ML 1.0037 1.0034 1.0032 1.0030 1.0029 1.0028
Viscosity 54.67 53.9 52.8 52.6 52.5 51.6
Osmolality 303 281 275 272 270 267
Assay (%) 96.8 96.0 95.7 95.3 94.8 94.4
Globule Size distribution D(10)=102.4
D(50)=464.8
D(90)=1976.6 D(10)=102.0
D(50)=462.8
D(90)=1974.4 D(10)=101.4
D(50)=462.4
D(90)=1974.3 D(10)=101.2
D(50)=462.1
D(90)=1973.6 D(10)=101.1
D(50)=461.8
D(90)=1972.6 D(10)=100.3
D(50)=461.4
D(90)=1970.4
PDI 0.316 0.319 0.320 0.323 0.330 0.336
Span 3.86 3.84 3.83 3.83 3.81 3.7
Zeta Potential -31.12 -30.05 -30.00 -28.54 -27.64 -27.12
Related Substance 0.42% 0.48% 0.54% 0.59% 0.64% 0.66%
Conclusion: -From Stability data at 40?C±2?C/25%RH of 1 M,2 M,3 M and 6 M at four different conditions upright with label & without label, inverted with label & Inverted without label we can conclude that there was no any significant change in the ophthalmic emulsion. So, we can conclude that product is stable in accelerated condition.

8.0 Conclusion:-
Delivery of drugs in eye using conventional drug delivery possesses several disadvantages like poor penetration in ocular surface and less corneal contact time using the approach of ophthalmic emulsion using Microfluidization technology possess several advantages like better permeability and higher corneal contact time can be obtained. Several studies show that preservative used in ophthalmic composition like Benzalkonium chloride result in eye irritation, To overcome this problem, present formulation is preservative free and microbial stable over the shelf life maintained using advance container closure system which allow filtered air to enter into container after application and avoid the microbial contamination.

The Experimental work results show that all the drug and excipients were compatible with each other. Microfluidizer Technology used to prepare ophthalmic emulsion was evaluated for pH, Viscosity, Globule Size distribution, PDI, Osmolality, Assay. The pH and viscosity of finished product was found in the range of 7.2 to 7.5 and 52 to 54 cps respectively. The Globule size distribution of d (10), d (50), d (90) data obtained which is around 102.4, 464.4, 1976.6 nm respectively and Zeta potential of ophthalmic emulsion was about – 30 to -32 mv. The stability data of 40?c±2?c/25%±5 RH condition was also found satisfactory. Physiochemical evaluation of optimized composition was found similar to innovator product.

9.0 References:-
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