A Thesis Entitled Development and Evaluation of a Nanomicellar Eye

Home , Eye drop, Papaverine

A Thesis

entitled

Development and Evaluation of a Nanomicellar Eye Drop Formulation of

Dexamethasone for Posterior Uveitis

Soohi Patel

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Pharmaceutical Sciences

Industrial Pharmacy

______Sai Hanuman Sagar Boddu, Ph.D., Committee Chair

______Jerry Nesamony, PhD, Committee Member

______Kenneth S. Alexander, PhD, Committee Member

______Patricia R. Komuniecki, PhD, Dean College of Graduate Studies

The University of Toledo

August 2014

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Development and Evaluation of a Nanomicellar Eye Drop Formulation of Dexamethasone for Posterior Uveitis

Soohi Patel

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Pharmaceutical Sciences

Industrial Pharmacy

The University of Toledo

August 2014

The overall objective of this study was to develop a clinically acceptable, mixed nanomicellar eye drop formulation of dexamethasone (DEXSOLV) for treating posterior uveitis. Nanomicelles were formulated using polyoxyl 40 stearate (P40S) and polysorbate

7/3 by weight. Transmission electron microscopy images revealed the spherical structure of micelles. DEXSOLV was found to be stable at 4oC and 25oC for up to 6 months. No irritation or redness was observed in the treated eyes as compared to the untreated control

iii rabbit eyes. Therapeutic concentrations of dexamethasone were observed in the rabbit retina and choroid following single and repeated topical administration. In conclusion, the nanomicelles of P40S and P80 could efficiently solubilize dexamethasone in their cores, resulting in a stable aqueous eye drop formulation. Using this eye drop formulation, dexamethasone concentrations could be maintained well above the minimum effective concentrations following topical administration. The proposed topical therapy would enhance patient compliance and minimize the side-effects associated with intraocular implants and intravitreal injections in posterior uveitis therapy.

Dedicated to my beloved family

Acknowledgements

I would like to express my deepest gratitude to Dr. Boddu for providing me an opportunity to be a part of his research group. This work would not have been possible without his valuable advice and excellent support all throughout my research. He provided me with all the resources and guidance needed to succeed with this project.

I would like to take this opportunity to thank Dr. Jerry Nesamony and Dr.

Kenneth S. Alexander for being a part of my defense committee. I would also like to thank them for their insightful comments and encouragement throughout my undergraduate and graduate school. I would like to specially thank Dr. Caren Steinmiller for serving as a graduate faculty representative for my thesis defense. I would like to thank Dr. Himanshu Gupta for helping me with the in vivo studies. I would also like to thank Dr. Wayne Hoss for supporting me financially throughout my graduate school.

I hugely extend my thanks to Chandrasekhar Garapati and Yangjie Wei for always helping me with my research and always being there, whenever I needed. I thank

Khyati Patel for helping me with her artistic skills. I would like to thank Yatri, Ruchin,

Khusbu, Hardik, Jay, Amrata, Drishti, Harish and Ruthwik for their cheerful support.

Finally, I would like to thank my wonderful parents (Sonal and Maullin Patel), my sweet brother (Munjal Patel), my jovial nani, and my entire family, for their financial and emotional support, without whom this all would not have been possible.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Tables ...... viii

List of Figures ...... ix

1. Introduction ...... 1

1.1. Anatomy of the eye ...... 3

1.1.1. Conjunctiva ...... 3

1.1.2. Cornea ...... 5

1.1.3. Aqueous Humor ...... 7

1.1.4. Iris ...... 10

1.1.5. Lens ...... 11

1.1.6. Vitreous Humor ...... 12

1.1.7. Sclera...... 12

1.1.8. Retina ...... 13

1.1.9. Optic Nerve ...... 14

1.2. Retinal Diseases ...... 15

1.2.1. Age-Related Macular Degeneration (AMD) ...... 15

1.2.2. Diabetic Retinopathy (DR) ...... 17

1.2.3. Diabetic Macular Edema (DME) ...... 19 vi

1.2.4. Proliferative Vitreoretinopathy (PVR) ...... 20

1.2.5. Uveitis ...... 21

1.2.6. Cytomegalovirus Retinitis (CMV) ...... 22

1.2.7. Glaucoma ...... 23

1.3. Challenges to ocular drug delivery ...... 24

1.4. Retinal drug delivery...... 26

1.5. Posterior penetration of topically instilled drugs ...... 29

2. Significance of Research...... 38

3. Development and Evaluation of a Nanomicellar Eye Drop Formulation of

Dexamethasone for Posterior Uveitis ...... 40

3.1. Abstract ...... 40

3.2. Introduction ...... 42

3.3. Materials and Methods ...... 45

3.4. Results and Discussion ...... 55

3.5. Conclusion ...... 73

4. Preclinical Evaluation of a Nanomicellar Eye Drop Formulation of Dexamethasone

for Posterior Uveitis Following Repeated Topical Administration ...... 74

4.1. Abstract ...... 74

4.2. Introduction ...... 75

4.3. Materials and Methods ...... 77

4.4. Results and Discussion ...... 79

4.5. Conclusion ...... 82

References ...... 83

vii

List of Tables

3.1 Solubility study of dexamethasone with increasing surfactant concentration ...... 56

3.2 Clarity test of 0.1% dexamethasone mixed nanomicellar formulation ...... 65

3.3 Artificial tear dilution test of 0.1% dexamethasone mixed nanomicellar

formulation ...... 66

3.4 Stability study over time ...... 67

3.5 Filtration efficiency test of 0.1% dexamethasone mixed nanomicellar

formulation ...... 68

3.6 Sterility validation test performed on MH Agar plates indicating the presence (+)

or absence (-) of microbial growth on days 0, 7, and 14 ...... 69

3.7 Ocular irritation study in rabbit using a blank mixed nanomicellar formulation ..71

4.1 Ocular irritation study in rabbit eyes following multiple drug loaded mixed

nanomicellar formulation ...... 80

viii

List of Figures

1-1 Structure of eye ...... 2

1-2 Parts of conjunctiva...... 5

1-3 Layers of Cornea ...... 7

1-4 Illustration of the trabecular meshwork conventional pathway ...... 9

1-5 Illustration of the uveoscleral non-conventional pathway ...... 10

1-6 Structure of retina ...... 14

1-7 Elimination of the administered dose through various pathways ...... 25

1-8 Drug delivery routes to the eye ...... 27

3-1 (A) DEXSOLV eye drops, (B) Structure of mixed nanomicelle ...... 55

3-2 Variation of the surface tension with surface concentration ...... 57

3-3 Transmission electron microscopy image of mixed nanomicelles ...... 58

3-4 Particle size and zeta potential curves of mixed nanomicellar formulation .... 60-61

3-5 Differential scanning calorimetry (DSC) thermogram ...... 62

3-6 (A) Calibration curve of dexamethasone, (B) Sample HPLC chromatogram of

dexamethasone ...... 63

3-7 In vitro drug release of dexamethasone from mixed micelles ...... 64

3-8 Sterility validation test performed on MH Agar plates indicating no bacterial

growth at 14 days ...... 70

3-9 Dexamethasone concentration in ocular tissues following a single topical

administration of DEXSOLV ...... 71

4-1 Treated rabbit eyes ...... 81

4-2 Dexamethasone concentration in ocular tissues following multiple administration

of DEXSOLV...... 81

Chapter 1

Introduction

Efficient drug delivery to the back of the eye remains a challenging task due to its unique anatomical and physiological barriers [1]. The external and internal structures of the eye are protected by different barriers that prevent the penetration of drugs in required concentrations. The structure of the eye is divided into two segments: anterior and posterior. The anterior segment consists of the cornea, iris/ciliary body, and lens [2]. The posterior segment mainly consists of the vitreous humor, retina, choroid, and optic nerve

(Fig. 1-1).

Figure 1-1: Structure of eye

A fully grown eye approximately weighs 7.5 g and has a volume of 6.5 ml. In a newborn the diameter of the eyeball is ~1.8 cm which gradually increases to about 2.54 cm in adulthood. A full size eye is attained at the age of thirteen. The eye can be considered as a photoreceptor with a shading pigment that facilitates the recognition of the light source direction [3]. Once the photoreceptor cells start growing in number, the transparent cells segregate into a transparent humor, which improves the refractive index of the eye and

helps in filtering colors [4]. The lens is formed due to an increase in the concentration of the crystalline proteins inside the transparent cells. The refractive power is further increased by the nontransparent iris and the transparent corneal layer, which are separated by the aqueous humor [5]. Light refraction is governed by the aqueous humor, lens, and the vitreous humor. The eyeball, except the cornea, is coated with the sclera, which is a white lining that protects the eye from injuries. The eye wall is composed of three layers: outermost, intermediate, and innermost layers. The outermost layer, also known as fibrous tunica, consists of the cornea, sclera, and corneoscleral limbus. The intermediate layer is pigmented tunica consisting of the choroid, ciliary body and iris. The innermost layer is nervous tunica consisting of the retina, photoreceptor cells, modulator cells, transmitter cells, and supporting cells.

1.1. Anatomy of the eye

1.1.1. Conjunctiva

The conjunctiva is a thin, transparent tissue that covers the visible part of the sclera and the inner surface of the eyelids. It is a mucous membrane composed of non-keratinized, squamous epithelium with goblet cells. Blood vessels, fibrous tissues, lymphatic channels are found in the epithelial layer. The conjunctiva is mainly divided into two parts: bulbar conjunctiva and palpebral conjunctiva (Fig. 1-2). Palpebral conjunctiva can be further divided into three parts: marginal conjunctiva, tarsal conjunctiva, and orbital conjunctiva.

The marginal conjunctiva is made up of stratified epithelium and it starts from the inter- marginal strips of the eyelid. It continues to the back of the eyelid up to a shallow groove 3

called the sulcus sub-tarsalis. The tarsal conjunctiva is adherent to tarsal plates and is highly vascular. It fully adheres to the entire tarsal plate. Through the transparent tarsal conjunctiva, the tarsal glands are visible as parallel yellow lining running in a vertical direction. The orbital conjunctiva is located over the Muller’s muscle in the upper eyelid and folds horizontally during eye movements. It covers the tarsal plate and fornix. The bulbar conjunctiva is divided into two parts: scleral conjunctiva and limbal conjunctiva.

The scleral conjunctiva is the thinnest and transparent conjunctiva with several visible blood vessels. The limbal conjunctiva is fused with the corneal epithelium and it covers the limbal region. The blood supply to the bulbar conjunctiva is maintained by the anterior conjunctival arteries and posterior conjunctival arteries. The blood supply to the palpebral conjunctiva is maintained by the marginal tarsal arcade and the descending branches of the peripheral tarsal arcade. The goblet cells present in between the conjunctival epithelial cells are responsible for secreting mucin that helps in nourishing the cornea. The secretion of mucin also helps in hydrating the bulbar and palpebral conjunctiva. The conjunctiva also contains mast cells, plasma cells, and lymphatic vessels

[6].

Figure 1-2: Parts of conjunctiva

1.1.2. Cornea

It is an outermost clear part of the eye that covers the anterior segment of the eye. The cornea is prone to infection due to the lack of blood vessels, which protects tissues. It receives nourishment from aqueous humor and tears [7, 8]. The cornea consists of a highly organized group of cells and proteins [6]. The focusing of incident light entering the eye is governed by the cornea and the crystalline lens. The cornea is composed of five different layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane, and

endothelium (Fig. 1-3). The epithelium is the outermost region of cornea comprising of nonkeratinized, stratified squamous epithelium, which is continuous with the bulbar conjunctiva [9]. It is nourished by the nutrients present in the tear fluids and is capable of absorbing oxygen. The surface epithelium prevents the passage of any alien material into the eye. The Bowman’s layer is a transparent sheet of tissue composed collagen that is located beneath the epithelium. Injury to this layer could result in scar formation, which might result in the loss of vision [9]. The stroma is located beneath the Bowman’s layer.

It is the largest portion of the cornea and comprises of ~90% of the total corneal thickness. It does not contain any blood vessels, but it is primarily made up of collagen and water. The strength and elasticity of the cornea are provided by the collagen. The

Descemet’s membrane is a basement membrane, located under the stroma. It is a strong sheet of tissue that acts as a barrier against infections. It is composed of collagen fibers and is regenerated after an injury [6]. The endothelium is the innermost layer of the cornea, which comprises of a single layer of transparent, nucleated cells that maintain the corneal transparency by balancing the fluids within the stroma. If the endothelial cells are destroyed by a disease, they do not regenerate and may lead to blindness or corneal edema.

Figure 1-3: Layers of cornea

1.1.3. Aqueous Humor

Aqueous humor is a clear fluid that separates of the anterior and posterior segments of the eye. It is formed by the ciliary epithelium and from the blood plasma through diffusion, ultrafiltration, and active transport [10]. Aqueous humor formation is an outcome of active secretion and follows a circadian rhythm in humans. The rate of secretion is higher in the morning as compared to the night. Sleep, age, retinal detachment, choroidal

detachment, and retinal diseases alleviate the rate in the production of aqueous humor. It is majorly composed of carbohydrates, organic and inorganic ions, proteins and amino acids, urea, carbon dioxide, oxygen, and water [11]. The aqueous humor leaves the eye via two pathways: the trabecular mesh-work and the uveoscleral pathway. In trabecular meshwork pathway, the fluid enters through the pupil and exits through the trabecular meshwork across the inner wall of the Schlemm’s canal into aqueous and episcleral veins

(Fig. 1-4) [12]. In uveoscleral pathway, the aqueous humor enters the connective tissue through the supraciliary space and exits through the sclera (Fig. 1-5) [13]. Due to the contraction and relaxation of the ciliary muscles, the aqueous humor outflow is altered.

Contraction of the ciliary muscles increases the trabecular outflow, while relaxation of the ciliary muscles decreases the trabecular outflow. It plays an important role in the maintenance of intraocular pressure [14].

Figure 1-4: Illustration of the trabecular meshwork conventional pathway

Figure 1-5: Illustration of the uveoscleral non-conventional pathway

1.1.4. Iris

The iris is circular in shape with a circular aperture in the center known as the pupil. It contains muscles which allow the pupil to dilate and constrict. It is located behind the cornea and in the aqueous humor. It controls the amount of light that enters the eye and adjusts the pupil opening. Through the iris sphincter and dilator muscles, the pupil can change its opening size [6]. The sphincter muscle comprises of a smooth muscle which is 10

located in the stroma on the posterior surface of the iris. The dilation with contraction of the iris is governed by the dilator muscles. In bright light, the iris constricts and reduces the size of pupil opening, which in turn restricts the amount of light entering the eye. In dim light, the pupil opening becomes larger and increases the amount of light entering the eye [15]. The color of the eye is determined by the iris. Based on the amount of pigmentation of the stroma, the color of an eye is determined. Independent of the color, all irises have the same stromal structure composed of melanocytes, collagen fibrils, non- pigmented cells, and a matrix containing hyaluronic acid. People with brown iris have a rich pigmented stroma, which increases the absorption of light. People with blue iris have poorly pigmented stroma which decreases the absorption of light. Throughout the stroma, there are several nerves and blood vessels. Also, there are myelinated and non-myelinated fibers in the stroma. The posterior surface of the iris, known as the posterior pigmented layer, is heavily pigmented. Whereas, the basal surface of the pigmented layer adheres to the anterior pigmented layer [9].

1.1.5. Lens

The lens is a transparent, biconvex structure that is suspended behind the iris and the pupil. It works with the cornea to focus the image onto the retina. The flexibility and elasticity of the lens helps in modifying its curvature to properly focus on the objects nearby or at a distance [9]. There is an inner nucleus and an outer cortex in the lens.

Being a basal lamina, the lens capsule is rich in collagen and other matrix proteins. The lens has a much lower oxygen concentration than the other parts of the body due to 11

insufficient blood supply [16]. The shape of the lens and its curvature is changed by the contraction of the ciliary muscles, which are attached to the lens. For near vision, the curvature of the lens becomes steeper, whereas for distant vision the curvature of the lens becomes flatter.

1.1.6. Vitreous humor

The vitreous humor is a jelly-like substance that comprises of almost 80% of the volume of the posterior segment of the eye. It is produced by the retinal cells. It fills up the space between the lens and the retina. Unlike the continuously replenished aqueous humor, vitreous humor is stagnant with composition similar to the cornea [17]. It comprises of water (99%), hyaluronic acid, inorganic salts, sugar, hyalocytes, ascorbic acid, and collagen fibrils [18]. The unwanted cellular debris from the vitreous humor is removed with the help of hyalocytes. The vitreous humor is viscous due to the non-branching of the collagen fibers with the hyaluronic acid. The vitreous humor transforms to a more liquid state with age and can detach from the back of the eye.

1.1.7. Sclera

The sclera is a tough, white portion of the eye that is opaque and fibrous in nature. It covers the entire eyeball except the cornea. Movement of the eye is governed by the muscles attached to the sclera [19]. It comprises of water (~68%), proteins (~3%), proteoglycans (~0.9%), elastin (~1-2%), and collagen (~28.8%) [20]. There are various subtypes of collagen in the sclera and each subtype acts in a unique manner. The visual 12

apparatus of the inner eye is supported by the robust collagen framework. The robust framework of the collagen in the sclera is supported by the amorphous components and elastin fibers consisting of microfibrillar. These elastin fibers are present in the inner layers of the stroma and are synthesized by scleral fibroblasts [21].

1.1.8. Retina

The retina is a multi-layered, light sensitive tissue that lines the innermost surface of the eye (Fig. 1-6). It is divided into the retinal pigment epithelium and neural retina. The retinal pigment epithelium is located between the neural portion of the retina and choroid.

The neural retina is an organized multi-layered membrane comprised of rods, cones, bipolar cells, and ganglionic cells [22]. The retina is comprised of millions of rod and cone cells, which are also known as photoreceptors. The photoreceptors are present in the macula, which is the smallest sensitive area in the center of the retina and mainly responsible for a clear central vision. The fovea is located in the center of the macula and is responsible for image sharpness. The incoming light is converted into electrical impulses by the cells in the retina. The optic nerve carries these electrical impulses to the brain, where they are interpreted as images.

Figure 1-6: Structure of retina

1.1.9. Optic Nerve

The optic nerve, also known as cranial nerve II, transmits the visual information from the retina to the brain. It is extended to the optic foramen from the eyeball. It is located within the orbit and is derived from the embryonic retinal ganglion cell. It can be classified into intraocular portion, intraorbital portion, intracanalicular portion, and intracranial portion. The optic nerve is composed of millions of retinal ganglion cells of a single retina. The retinal ganglionic cell axons and the Portort cells make up the optic nerve fibers. There are three meningeal layers covering these optic nerve fibers: dura,

arachnoid, and pia mater. Sensory modality of the vision is carried by the special somatic afferent in the optic nerve [23].

1.2. Retinal diseases

Diseases affecting the posterior segment such as age-related macular degeneration

[AMD], diabetic retinopathy [DR], diabetic macular edema [DME], proliferative vitreoretinopathy [PVR], uveitis, and cytomegalovirus [CMV] require immediate attention in order to prevent the loss of vision [24-26].

1.2.1. Age-Related Macular Degeneration (AMD)

AMD remains one of the major vision-threatening ocular diseases among the elderly population [27]. It occurs primarily in two forms: dry AMD (non-exudative) and wet

AMD (exudative or neovascular). Age is considered one of the major risk factors among

AMD patients. The mean age for dry AMD is 56.8 years, while the mean age for wet

AMD is 70.5 years. Apart from age, the other risk factors for AMD include smoking, obesity, race, gender and family history [28]. Dry AMD occurs more commonly than does wet AMD. It is characterized by slow breakdown of photoreceptors, RPE and choriocapillaries, leading to blur in the central vision. At the onset, this disease affects only one eye with the formation of small to medium-sized drusen (yellow deposits formed under the retina) that gradually spread to the other eye and finally result in vision loss [29, 30]. The exact cause, mechanism and treatment of dry AMD are not clear. Some

studies suggest that specific high-dose formulations containing antioxidants, zinc and vitamin supplements can delay the advancement of dry AMD considerably.

Wet AMD is characterized by the growth of abnormal blood vessels behind the retina.

These blood vessels are often fragile, leading to leakage of blood beneath the macula.

Other pathophysiological processes associated with wet AMD include drusen deposits, disruption of Bruch’s membrane and degeneration of RPE, leading to complete loss of vision. Wet AMD patients are frequently characterized by high levels of vascular endothelial growth factor (VEGF), which is a potent mitogen of vascular endothelial cells. VEGF is a homodimeric (compound containing two identical molecules) glycoprotein known to induce angiogenesis and vascular permeability. Various isoforms of VEGF have been identified in humans. The major isoforms include VEGF121,

VEGF165, VEGF189, and VEGF206, and minor isoforms include VEGF145, VEGF148,

VEGF162, VEGF165b and VEGF183. Of all isoforms, VEGF165 is abundantly expressed and is primarily responsible for angiogenesis and endothelial cell growth [31].

Currently, wet AMD is treated using photodynamic therapy, laser treatment and intravitreal injection of anti-angiogenic agents. Photodynamic therapy involves the intravenous infusion of the light-activated drug, vertiporfin [32]. This agent has a tendency to accumulate in abnormal blood vessels in the retina. Following accumulation, a low energy laser beam is used to activate the drug and destroy the abnormal blood vessels. In laser treatment, the leaky blood vessels are sealed using a high-energy laser beam [33]. This improves the vision only for a short period of time. Intravitreal injection 16

of anti-VEGF agents have revolutionized the treatment of wet AMD. Anti-VEGF agents like ranibizumab, pegaptanib sodium and bevacizumab are widely administered as intravitreal injections. These agents act by blocking various isoforms of VEGF that are responsible for angiogenesis and vascular permeability [34]. Pegaptanib sodium has limited clinical success, as it does not bind all isoforms of VEGF-A [35]. Though ranibizumab binds all VEGF isoforms, its clinical success is limited by inefficient delivery strategies. Currently ranibizumab is administered by frequent, painful intravitreal injections. Clinically efficacious approaches to inhibiting VEGF-A for AMD treatment require a safe and less invasive delivery system, which is yet to be realized

[35].

1.2.2. Diabetic retinopathy (DR)

DR is one of the most common causes of blindness in developed countries, and each year

DR causes more than 8000 people to lose their vision in the United States alone. DR involves hyperglycemic-mediated damage to small blood vessels in the retina. DR affects

70% of all people with diabetes over their lifetimes and 60% of people who have had diabetes mellitus for more than 15 years. The first sign of histological damage, before clinical symptomology, includes basement membrane thickening and increased retinal blood flow that is secondary to tissue hypoxia-mediated vasodilation [36]. Hypoxia also stimulates the release of growth factors that promote vascular proliferation in the retina.

This neovascularization is dysfunctional and inhibits the retina’s intrinsic ability to detect light. DR occurs in various forms. Background retinopathy is the common form and can

be present with normal vision. Small micro-aneurisms and hemorrhages appear as cotton wool spots in an eye examination. These defects preferentially target the portion of retina that is temporal to the macula, but may advance medially, causing frank maculopathy.

This is the predominant cause of vision loss among diabetic retinopathy patients.

Maculopathy is further subcategorized based on predominating pathology such as lipid exudation, diffuse edema, or ischemia [37, 38].

The main treatment for DR involves laser eye surgery or photocoagulation that creates small burns on the abnormal blood vessels in the retina. This process prevents leaking of blood vessels and terminates the growth of abnormal and fragile vessels. Vitrectomy is also used to prevent hemorrhage and to repair retinal detachment. Corticosteroids, agents that prevent the proliferation of blood vessels, angiotensin-converting enzyme inhibitors and growth hormone, have recently been investigated in DR treatment [39, 40]. Park et al. [41] evaluated the effect of kringle 5 (K5) loaded nanoparticles as angiogenic inhibitors on retinal inflammation and for treatment of vascular leakage and neovascularization in a DR model. K5 is a proteolytic fragment (80–amino acid peptide) of plasminogen with a potent inhibitory effect on endothelial cell growth [42]. Intravitreal injection of nanoparticles resulted in high expression levels of K5 in the inner retinas of rats and reduced the retinal vascular leakage and retinal neovascularization without toxicity.

1.2.3. Diabetic macular edema (DME)

DME primarily affects people with a history of diabetes mellitus. It is characterized by swelling of the retina within the macula, which gradually results in leakage of fluids from blood vessels and breakdown of blood-retinal barrier [43]. The macula is a small structure at the center of the retina that is rich in cones and specialized nerve endings.

Apart from diabetes mellitus, hypertension also leads to an increase in hydrostatic pressure within blood capillaries, resulting in expulsion of fluid into the retina. DME can be broadly classified into two types: focal or non-cystoid DME and diffuse or cystoid

DME. Noncystoid edema is caused by small aberrations in retinal blood vessels followed by intraretinal leakage. Cystoid edema is associated with the formation of microcysts and dilation of retinal capillaries. Focal or grid lasers are currently being used in the treatment of DME. In focal laser treatment, leaky blood vessels are sealed while grid laser treatment is applied to dilated retinal capillaries [44, 45]. Failure of patients to completely recover following laser treatment has promoted vision scientists to develop alternative strategies. More insight into the pathophysiological processes of DME reveals that the increase in retinal capillary permeability following the breakdown of blood retinal barrier is partially mediated by VEGF [46]. A recent report by Nauck et al. [47] suggests that corticosteroids significantly inhibit the expression of the gene responsible for production of VEGF [47]. This finding has generated considerable interest regarding the use of corticosteroids in the treatment of DME. Nanoparticulate systems of corticosteroids are being investigated for sustained delivery of drugs to the retina/choroid following episcleral administration [48].

1.2.4. Proliferative vitreoretinopathy (PVR)

PVR is the most common complication associated with retinal detachment surgery. It is characterized by scar formation and proliferation of cells in vitreous and retina [49, 50].

“Proliferative” represents cell proliferation, and “vitreoretinopathy” represents problems involving vitreous and retina. PVR can be divided into multiple categories based on the inflammation of retina (focal, diffuse, sub-retinal, circumferential and anterior displacement) and the location of the scar tissue (anterior and posterior). The therapeutic options available for modifying the healing response after retinal detachment are limited

[51]. Currently, PVR is managed by surgery. Patients remain on adjunctive treatment after surgery to avoid relapses. A combination of 5-fluorouracil and low molecular weight heparin primarily aids in the treatment of patients who are at risk of developing

PVR following retinal reattachment surgery [52]. Proliferative retinopathy secondary to diabetic neovascularization is present later in the disease course of uncontrolled hyperglycemia. Because retinal capillaries are fenestrated, they have a tendency to leak and are implicated in retinal detachment. This happens most commonly in the setting of fibrosis with neovascularization, also called retinitis proliferans. Zhang et al. [51] demonstrated delivery of drugs to the posterior pole using aerosolized nanoparticles in the gas phase of vitrectomy. This method can also be used in the delivery of antimetabolites to modulate PVR, antimicrobial agents for endophthalmitis, antiangiogenic compounds for vasoproliferative disorders, and corticosteroids to the retina and choroid.

1.2.5. Uveitis

Uveitis refers to inflammation occurring in the middle layer of eye known as uvea, the vascular layer between the retina and the sclera. The exact cause of uveitis is not clear, but studies indicate that viral (mumps and herpes), fungal (histoplasmosis) and bacterial

(toxoplasmosis) infections might play an important role [53]. Based on the structures affected, uveitis can be categorized into several forms: anterior, intermediate, posterior and pan-uveitic forms. Anterior uveitis is the most common form of uveitis, affecting the iris and anterior chamber, while posterior uveitis affects primarily the retina and choroid.

Intermediate uveitis is characterized by inflammation in the vitreous cavity, while panuveitis affects all layers of uvea [54]. Uveitis is treated using glucocorticoids administered as eye drops and topical cycloplegics. In some cases, posterior subtenon triamcinolone acetate is administered to reduce the swelling of eye [55, 56]. Sakai et al.

[57] studied the therapeutic effects of betamethasone phosphate loaded in PLA and PEG- block-PLA copolymers for treatment of experimental autoimmune uveoretinitis (EAU) model in Lewis rats. They observed accumulation of Cy7-stealth nanoparticles in inflamed eyes of rats for a three-day period. Stealth-nanosteroids (100 μg of betamethasone phosphate) reduced the clinical scores of rats within one day and maintained the effect for two weeks following systemic administration.

1.2.6. Cytomegalovirus retinitis (CMV)

CMV is a viral infection characterized by inflammation of the retina. Cytomegalovirus is a Greek term that denotes a large cell virus belonging to the family of human herpes virus. CMV progressively leads to retinal detachment and complete blindness. The virus has the ability to remain latent inside the body for a long duration. This disease primarily affects HIV patients with T cell deficiency and in whom CD4 count is less than 100.

Earlier statistics suggest that CMV retinitis occurs in one-third of patients suffering from

AIDS and is the major cause of blindness in AIDS patients [58]. CMV resides in various body fluids such as saliva, blood, urine, cervical secretions and semen. The virus spreads upon blood transfusion, sexual contact, breast-feeding, and organ transplantation. CMV can be treated successfully with a combination of medications that act specifically against the virus and thus protect the immune system. Highly Active Anti-Retroviral Therapy

(HAART) has significantly reduced CMV retinitis problems in developed countries [59].

Antiviral agents like cidofovir, ganciclovir and foscarnet have also been widely employed in the treatment of CMV retinitis. The antiviral drugs act only as virustatic and are not virucidal. Due to short vitreal half-life, most antiviral drugs are administered frequently in the form of painful intravitreal injections. Though implants overcome the disadvantages associated with intravitreal injections, surgical procedure and risk of drug precipitation due to poor solubility cause side effects [60]. Furthermore, implants can also cause astigmatism or vitreous hemorrhages [61]. Polymeric nanoparticles can serve as an alternative for chronic ocular diseases requiring frequent drug administration, such as

CMV.

1.2.7. Glaucoma

Glaucoma is a progressive optic nerve disorder characterized by elevated intraocular pressure (IOP) and loss of retinal ganglionic cells. The optic nerve (or cranial nerve II) is composed of retinal ganglionic cell axons and helps to transmit visual information directly from the retina to the brain. Statistics reveal that approximately 2.5 million people are affected by glaucoma in United States [62]. Early diagnosis and treatment play a vital role in halting the progression of glaucoma. Obstruction of the outflow of aqueous humor from the anterior segment causes glaucoma. Aqueous humor is secreted by the non-pigmented epithelium of the ciliary body, which flows from the posterior to the anterior chambers before draining through the trabecular meshwork via Schlemm’s canal

[63]. IOP of a normal eye is 21 mmHg, which is altered by variations in inflow and outflow of aqueous humor [64]. Glaucoma can be broadly divided into two categories: primary open angle glaucoma (POAG) and angle closure glaucoma (ACG). POAG results from the clogging of the trabecular meshwork canal, resulting in rise of IOP [65,

66]. In ACG, the iris and lens restrict the movement of fluid between the chambers. As a result of this pressure, the iris pushes the trabecular meshwork, building up pressure inside the eye [67].

Currently glaucoma is treated with eye drops, laser treatment and surgery. Various drug categories currently employed in glaucoma treatment include: prostaglandin analogs, which increase the uveoscleral outflow of aqueous humor [68]; beta-adrenergic receptor 23

antagonists, which decrease aqueous humor production by the ciliary body; alpha2- adrenergic agonists, which decrease aqueous production and increase uveo-scleral outflow [69]; parasympathomimetics, which increase outflow of aqueous humor through trabecular meshwork [62]; and carbonic anhydrase inhibitors, which lower secretion of aqueous humor [70]. Although several efficacious drugs exist for reducing the IOP, their potential is limited by the lack of suitable delivery systems that can deliver the drugs locally in a sustained fashion. In some patients who continue to show progression regardless of IOP reduction, complementary neuroprotective approaches are desired.

However, most of the potential neuroprotective agents are not suitable for oral delivery or eye drop formulations and thus require the development of new technologies aimed at increasing clinical efficacy by offering multiple delivery options for several months to the retina and optic nerve [71, 72].

1.3. Challenges to ocular drug delivery

Drug delivery to the eye is challenging due to its unique ocular anatomy and physiology.

Most anatomical and physiology barriers protect the eye from foreign particles, including drug molecules. These barriers vary based on the route of administration such as topical, systemic, and injectable. For the treatment of anterior segment diseases, topical administration of drugs is the most preferred route. Following topical administration, the absorption of a drug occurs through the corneal or non-corneal pathways (Fig. 1-7). The absorption of the drug through the corneal pathway enters the anterior ocular tissues,

whereas the absorption of the drug through non-corneal pathway results in systemic drainage via the nasolacrimal duct [73, 74]. The pre-

Figure 1-7: Elimination of the administered dose through various pathways.

corneal loss factors such as lacrimation, solution drainage, tear dilution, tear turnover, non-productive absorption, impermeable corneal epithelium, and transient residence time in the cul-de-sac, results in a poor bioavailability of drugs from ocular dosage forms. Due

to these anatomical and physiological constraints, less than 7-10% of the administered dose is absorbed into the eye [75].

1.4. Retinal drug delivery

Posterior segment eye diseases present unique anatomical, physiological, and biochemical barriers resulting in the failure of the conventional dosage forms such as eye drops, ointments and suspensions. The retina and choroid are the target sites for most of the posterior segment diseases. Following topical administration, static barriers (corneal layers, blood aqueous and blood-retinal barriers) and dynamic barriers (choroidal and conjunctival blood flow, lymphatic clearance, and tear dilution) prevent the drug from reaching the retina. Only about 1/100,000th of the drug observed in tear fluids reaches the retina and choroid [76]. Common approaches to the treatment of posterior segment diseases include, but are not limited to, systemic and intraocular injections and implants.

Systemic drug delivery results in inadequate retinal concentrations and severe systemic adverse effects. Intravitreal injection involves the administration of drug into the vitreal cavity with a 30 gauge needle (Fig. 1-8). Compared to systemic administration, higher concentration of drug is observed in vitreous humor and retina following intravitreal injection.

Though intravitreal administration delivers a high concentration of drugs to the retina, the inherent potential side effects like increased intraocular pressure [77], hemorrhage [78], cataract [79], and endophthalmitis [80] lead to complications limiting long-term therapy 26

[61]. Moreover, the chronic nature of retinal diseases requires multiple injections, which are associated with risk of vitreous hemorrhage, retinal detachment, and cataract progression. Implants have overcome many of the disadvantages associated with intravitreal injections; however, the surgical procedure and risk of drug precipitation may result in undesirable effects [81]. Periocular route is also widely used for the posterior segment delivery of drugs. It includes various routes of administration such as peribulbar route, retrobulbar route, sub-tenon, and sub-conjunctival route (Fig. 1-8). Periocular route is considered to be a promising route. Although, following periocular injection, there are some complications observed such as cataract, high retinal concentrations, high vitreal concentrations, corneal decompensation, hyphema, and an increase in intraocular pressure [82].

Figure 1-8: Drug delivery routes to the eye 27

A non-invasive topical drug delivery system in the form of eye drops would circumvent most of these problems and enhance patient compliance. Administration of drugs in the form of eye drops for retinal diseases has several advantages; it allows self- administration, localized therapeutic effect, a non-invasive and painless mode of drug administration, and high patient compliance. New treatment modalities to facilitate the topical delivery of drugs are under development. There are currently no marketed topical formulations for posterior segment diseases, with only a few compounds that are undergoing testing in clinical trials. Topical eye drops have the potential to reduce the side-effects and improve patients’ quality of life and patient compliance.

Pharmacokinetic studies have proven repeatedly that the transfer of a drug from an eye drop into the retina or vitreous humor is limited. This is mainly because of the long diffusional distance from the application site to the retina and the above mentioned dynamic barriers [83]. However, during the last decade, some investigators have documented the ability of small molecules to achieve therapeutic concentrations in the retina/vitreous humor following topical application [84-89]. Drug delivery to the back of the eye has become the topic of intense research among ophthalmologists and pharmaceutical scientists due to its ability to reduce unwanted complications resulting from intravitreal injections and to enhance patient compliance. The previous notion that drugs applied topically do not penetrate to the back of the eye is currently being reassessed.

1.5. Posterior penetration of topically instilled drugs

Despite the efforts of pharmaceutical scientists worldwide, drug delivery to the back of the eye remains challenging due to the unfavorable anatomy, physiology and biochemistry of the eye. Much of the current research is directed towards the development of effective drugs that penetrate into the posterior tissues following topical administration. There has been a limited number of studies published in the literature supporting the penetration of drugs into the vitreous and retina after topical instillation in rabbits [88]. However, the mechanism of penetration still remains contentious. Some studies claim the direct penetration of drugs via corneal or non-corneal pathways, while several others support the drug being absorbed into the blood stream across the conjunctiva or nasolacrimal duct and then penetrating back into the ocular tissues of both eyes by crossing the blood-retinal barrier. In this section, we have attempted to summarize the research articles supporting the retinal penetration of drugs following topical administration.

Ahmed and Patton [90] studied the ocular distribution and disposition of topically applied timolol and inulin following corneal and non-corneal absorption in New Zealand Albino male rabbits. For timolol, in the presence of corneal access, the rank-order in terms of concentration was conjunctiva> cornea>sclera> iris-ciliary body >aqueous humor> lens > vitreous humor, from greatest to lowest concentration. With corneal access blocked, the aqueous humor and corneal drug levels dropped drastically, and concentrations in

intraocular tissues such as iris-ciliary body, lens and vitreous were low. The concentration of timolol in the sclera and conjunctiva remained similar for the corneal and non-corneal routes. A similar pattern was observed with inulin. This study demonstrated the absorption of drugs into posterior tissues via the corneal and non- corneal routes. The same group also evaluated the ocular distribution and disposition of topically applied timolol and inulin following the corneal and non-corneal absorption with time. The authors identified that conjunctival absorption is responsible for up to

40% of the absorbed amount of inulin in the eye, and both corneal and non-corneal absorption routes resulted in vitreal concentrations of inulin and timolol [91].

Acheampong et al. [84] studied the distribution of brimonidine (α2-adrenergic agonist) into anterior and posterior ocular tissues following administration of single or multiple doses of 0.2 or 0.5% brimonidine tartrate solution in one or both the eyes of monkeys or to a single eye in rabbits. [14C]-brimonidine was found to be rapidly absorbed into the cornea and conjunctiva and distributed throughout the eye, and the vitreous humor concentration of brimonidine was found to be 82 ± 45 nM. Similar results were observed in rabbits, confirming the ability of brimonidine to reach the back of the eye at nanomolar concentrations relevant in neuroprotection models.

A study by Osborne et al. [87] concluded that topically applied Betoptic(R) (0.5% betaxolol) in rabbit or rat eyes reach the retina and can counteract the detrimental effects caused by ischemia/reperfusion or N -methyl- d -aspartate (NMDA)-induced insults. In a different study, the same group reported the neuroprotective activity of flunarizine in 30

rabbit and rat retinas [92]. Kent et al. [93] reported the vitreous levels of brimonidine (< 2 nM concentration) following topical application of Purite 0.15% (b.i.d. or t.i.d.) for 2 weeks in patients scheduled for pars plana vitrectomy.

The penetration into the ipsilateral posterior retina–choroid of nipradilol, a α1, β-blocker with a nitric oxide donative action, was studied in rabbits, and its effect on N-methyl-D- aspartate (NMDA)–induced retinal damage was studied in rats. [14C]-nipradilol (1%,

100μL, 1.5 MBq [41 μCi]/dose) was topically instilled twice daily for a week and resulted in effective concentrations of nipradilol in the posterior segment (vitreous: 4.3 ±

0.4 and retina-choroid: 318.6 ± 42.9ng.equivalent/gm). Also, nipradilol effectively suppressed the NMDA-induced retinal damage in rats [94]. The same group later studied the route of penetration of topically instilled nipradilol into the ipsilateral posterior retina

[95].

Palanki et al. [96] have developed and tested several potent benzotriazine inhibitors for targeting VEGFr2, Src, and YES kinases. These ester analogs exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser- induced choroidal neovascularization model following topical administration. The ocular distribution and excretion of tritium-labeled difluprednate ((3) H-DFBA) ophthalmic emulsion 0.05% after single or repeated topical instillation was studied in pigmented rabbit eyes. (3)H-DFBA concentrations were observed in various anterior segment and posterior segment tissues, including anterior retina/choroid (273 ngeq/g) and posterior

retina/choroid (59 ngeq/g), suggesting the effectiveness of topical therapy in posterior segment diseases [97].

Tissue distribution and epithelial penetration of radiolabelled moxaverine, a papaverine– hydrochloride derivative, were studied in rabbits following systemic and topical administration. High concentrations of moxaverine were found in the cornea, conjunctiva

(anterior part) and retina (posterior part) with topical administration. Low plasma levels of moxaverine were attributed to the lipophilic nature of moxaverine [98]. Similarly,

Baklayan et al. studied the ocular distribution of 14C-bromfenac in rabbits following topical application. Peak concentrations in the aqueous humor and most ocular tissues including cornea, conjunctiva and sclera were observed in the first 2 hours. However, a much lower concentration was found in the posterior segment (retina) [99].

Eye drop formulations consisting of cyclodextrin-drug complexes are widely studied for retinal delivery following topical application. The relative efficiencies of topical and systemic absorption of dexamethasone-cyclodextrin complex were examined in rabbits.

The systemic absorption following topical application to the eye was compared with intravenous and intranasal administrations. With topical administration, dexamethasone concentration in the retina of treated rabbits was found to be 33 ± 7 ng/g, while 14 ± 3 ng/g was observed in the control eye. Topical, intranasal and intravenous routes resulted in similar systemic levels. The authors concluded that the topical absorption plays a significant role in delivering dexamethasone to the retina. Approximately 60% of the

dexamethasone concentration in the retina resulted from topical administration, with the rest coming from systemic administration [100].

Loftsson et al. [101] studied the retinal penetration of dexamethasone upon topical application of dexamethasone/ɣcyclodextrin microparticles with a mean particle diameter of 20.4±10.3 µm. After 2 hours, the vitreal concentration of dexamethasone was found to be 29 ± 16 ng/g, with 86% of drug reaching the vitreous via direct penetration. The retinal concentration was found to be 57 ± 22 ng/g (49% via direct penetration).

Microparticles made of randomly methylated b-cyclodextrin also resulted in similar vitreal (22.6 ± 9 ng/g) and retinal (66 ± 49 ng/g) concentrations. The same group also studied the safety and efficacy of cyclodextrin microparticles in nineteen diabetic macular edema (DME) patients. Dexamethasone-cyclodextrin microparticles in the form of eye drops were administered three or six times/day for 4 weeks and then observed for

4 weeks without treatment. The eye drop formulation was well tolerated and an improvement in the visual acuity was observed, with a decrease in central macular thickness [102].

The physicochemical properties of drug substances such as lipophilicity, water solubility, and molecular size play an important role in crossing the eye’s biological barriers and reaching the retina. For example, dorzolamide (324 Da) and brinzolamide (383 Da) are carbonic anhydrase inhibitors used in the treatment of primary open angle glaucoma.

Dorzolamide and brinzolamide have similar molecular weights; however, they differ in lipophilicity and water solubility. Topical application of dorzolamide resulted in a 33

significant improvement in blood flow to the retina and optic nerve head compared to brinzolamide [103]. The variation in the pharmacological effect is mainly due to the ability of dorzolamide to better penetrate the ocular barriers and reach the posterior segment of the eye in a higher concentration. The ocular pharmacokinetics of Trusopt

(dorzolamide hydrochloride ophthalmic solution, 2%) and Azopt (brinzolamide ophthalmic suspension, 1%) were compared in rabbits upon single and multiple topical dosing. After a single dose, the area under the curve (AUC0-24h) for dorzolamide in the aqueous humor, anterior sclera, posterior sclera, anterior retina, posterior retina, anterior vitreous, and optic nerve was 2-, 7-, 2.6-, 1.4-, 1.9-, 1.2-, and 9-fold higher than those of brinzolamide. Cmax of dorzolamide was 2-5 times higher than brinzolamide’s. A similar trend was observed after multiple dosing, however; statistically higher concentrations of dorzolamide were only obtained in the aqueous humor, vitreous humor, and optic nerve

[104]. The log D values of dorzolamide and brinzolamide at pH 7.4 are 1.72 and 6.6, respectively. For corneal penetration, the optimum log D value should range between 2.0 and 3.0. The higher concentrations of dorzolamide in various ocular tissues can be attributed to the better penetration and higher concentration-dependent flux of the drug across the cornea. In a different study, Jansook et al. developed dorzolamide self- assembled microparticle suspension using 18%w/v γ-cyclodextrin and 0.5%w/v of hydroxyl propyl methyl cellulose. Maximum drug concentration in aqueous humor was reached in 4 hours, along with its maximum availability in the posterior segment [105].

The ocular bioavailability and retinal concentrations of hesperidin and hesperetin were compared following intravenous and topical administration in rabbits. Hesperidin is 34

an aglycone form of hesperetin. Intravenous administration failed to demonstrate any detectable levels of hesperidin/hesperetin in ocular tissues, while topical administration produced significant concentrations of hesperidin/hesperetin in ocular tissues 1 and 3 hours post-dosing, with hesperetin concentrations higher than hesperidin concentrations.

Furthermore, benzalkonium chloride improved the hesperetin levels in the posterior section of eye [106]. In situ gelling polymers such as gellan gum also play a significant role in enhancing the permeation of drugs into the retina. The ocular pharmacokinetics and tissue distribution of aesculin, a coumarin glucoside, after topical administration in rabbit eye, were found to be significantly higher in the presence of deacetylated gellan gum. This is due to longer precorneal contact times of in situ gels compared to conventional eye drops [107].

Nanonosystems, such as nanoparticles, mixed micelles, animations, nano suspensions, liposomes, and dendrimers, are slowly making their presence felt in the complex area of ocular drug delivery. Nanocarriers have the potential to improve the efficacy and ocular bioavailability of drugs by overcoming diffusion barriers. Recently, Velagaleti et al.

[108] have developed nanomicellar formulations of vitamin E TPGS and octoxynol-40 for voclosporin, a next generation calcineurin inhibitor. Ocular distribution studies following topical application of nanomicellar formulations in rabbits revealed a significantly higher concentration of voclosporin in the posterior segment tissues such as retina and choroid, with vitreous humor concentration below detectable limits. The same mixed nanomicellar formulation also resulted in therapeutic concentrations of dexamethasone in the rabbit choroid/retina (~50 ng/g tissue) following topical application 35

[109]. However, the mechanism by which nanomicelles deliver the drug to the retina following topical administration is not well understood. As stated by Mitra, entrapping the hydrophobic drug within the hydrophilic mixed micelles helps the drug to evade the clearance mechanisms of the eye.

More recently, there has been a surge in the use of large protein and peptide molecules in treating retinal diseases. However, they fail to penetrate the inner eye following topical application, largely due to their large molecular weight of 50-150 kDa. Use of penetration enhancers (e.g. sodium caprate) aided in the permeation of topically applied antibody fragments such as scFv, a protein of ~28 kDa [110]. However, the use of penetration enhancers to enhance the transcorneal transport has resulted in ocular toxicity and loss in the integrity of inter-epithelial tight junctions. Currently, ophthalmologists are left with no options other than administering protein molecules via intravitreal injections, which are highly invasive and associated with patient non-compliance. Topical delivery of antibody-based therapeutics would minimize the side-effects resulting from intravitreal injections, and this would be a major innovation in ophthalmology [111]. Furrer et al. studied the pharmacokinetics of ESBA105 (26 kDa), a potent scFv directed against tumor necrosis factor. Pharmacokinetics following topical and intravenous administration were compared. Systemic exposure after topical administration is 25000-fold lower than the intravenous administration, whereas exposure to vitreous humor (half-life 16-24h) is higher via topical route than through intravenous injection. ESBA105 is absorbed and distributed to all compartments of the eye with low systemic drug exposure [112].

The studies discussed above clearly demonstrate the ability of topically administered eye drop formulations to deliver drugs to the retina and exert pharmacological effects. It is noteworthy that most of these studies have suggested drug absorption via the transconjunctival/transcleral route (conjunctiva sclera choroid retina).

Chapter 2

Significance of Research

Common approaches to the treatment of posterior segment diseases include topical administration, systemic drug delivery, intravitreal injections, and intraocular implants [113]. Systemic drug delivery results in inadequate retinal concentrations and severe systemic adverse effects [114]. Intravitreal administration delivers a high concentration of drugs to the retina and thus the inherent potential side effects like increased intraocular pressure, hemorrhage, cataract, and endophthalmitis lead to complications limiting long-term therapy. Moreover, the chronic nature of retinal diseases requires multiple injections, which are associated with risk of vitreous hemorrhage, retinal detachment, and traumatic cataract [115]. Intraocular implants have overcome many of the disadvantages associated with intravitreal injections; however, the surgical procedure and risk of drug precipitation may result in undesirable effects [116].

To overcome the limitations related to the current approaches of drug delivery for the treatment of posterior eye diseases, a non-invasive topical drug delivery system in the form of eye drops would be a better alternative which minimizes the side-effects associated with intravitreal injections and intraocular implants. Topical administration of 38

drugs in the form of eye drops is a widely accepted route of administration because it is convenient, allows self-administration, localized therapeutic effect, a non-invasive and painless mode of drug administration, and has a relatively high patient compliance.

Until recently, there was limited evidence from large clinical trials that demonstrated the usefulness of pharmacotherapy compared to laser therapy or other vitreoretinal surgical techniques for the treatment of retinal diseases. This paradigm has shifted in the last decade, with strong evidence demonstrating superior efficacy of corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) compared with previous therapy [117-119]. Corticosteroids are most often used to treat uveitis, despite the availability of immunosuppressive therapies. However, systemic corticosteroids are intolerant or resistant for many patients. Topical corticosteroids administered in the form of eye drops are better tolerated by the patients as compared to systemic corticosteroids.

There is a disadvantage associated with the use of topical corticosteroids because typically they are only effective for anterior uveitis [120]. The synthetic anti- inflammatory glucocorticoid, dexamethasone, is widely used for the treatment of serious inflammatory diseases, such as cerebral edema, allograft rejection and systemic lupus erythematosus. Recently, use of dexamethasone has been explored in the treatment of retinal diseases such as macular edema and uveitis [114].

The aim of this study is to prepare a novel mixed nanomicellar eye drop formulation as a promising carrier for dexamethasone using Polysorbate 80 (P80) and

Polyoxyl–40–Stearate (P40S) as surfactants. The drug loaded mixed nanomicellar formulation was prepared and evaluated. 39

Chapter 3

Development and Evaluation of a Nanomicellar Eye Drop Formulation of Dexamethasone for Posterior Uveitis

3.1. Abstract

80 (P80), which are approved by US FDA for ocular use. The nanomicellar formulation was characterized for critical micellar concentration, solubility of dexamethasone, pH, particle size, zeta potential, morphology, differential scanning calorimetry, in vitro drug release, clarity, stability, filtration efficiency, and sterility. Ocular tolerance and the tissue drug distribution of dexamethasone were assessed in rabbits following single topical administration. Nanomicellar formulation of dexamethasone (0.1%) was successfully developed and characterized with an optimized composition of P40S/P80 = 7/3 by weight. Transmission electron microscopy images revealed the spherical structure of micelles. DEXSOLV was found to be stable at 4oC and 25oC for up to 6 months. No irritation or redness was observed in the treated eyes as compared to the untreated control rabbit eyes. Therapeutic concentrations of dexamethasone were observed in the rabbit

retina and choroid following single topical application. In conclusion, the nanomicelles of

P40S and P80 could efficiently solubilize dexamethasone in their cores, resulting in a stable aqueous eye drop formulation.

3.2. Introduction

Uveitis, an autoimmune disease of the eye, is responsible for up to 10% of all blindness in the United States, with approximately 30,000 new cases diagnosed each year. Uveitis patients are estimated to account for more than 5 million eye clinic visits per year, while the care of these patients in the US is estimated to cost over $117 million annually.

Estimates also indicate that each year, 17.6% of patients with active uveitis experience transient or permanent loss of vision, 12.5% develop glaucoma, and 32% develop at least one complication during the course of a year. This inflammatory, Th1-mediated disease mainly occurs in the 20-50 year age group and can affect one or both eyes [121-123].

In uveitis, damaging chronic inflammation can occur in either the front of the eye

(anterior uveitis), the back of the eye/retina (posterior uveitis or choroiditis), the ciliary body (intermediary uveitis or cyclitis) or all layers of uvea (panuveitis). Treatment of posterior and panuveitis is challenging, as the anatomical and physiological properties that effectively protect the eye hinder efficient absorption of drugs [124]. Corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressants have shown beneficial effects in uveitis treatment [125]. However, delivery of these drugs to the retina is challenging, and ophthalmologists are left with no options other than administering them locally via implants and intravitreal injections, which are highly invasive and are associated with side effects and patient non-compliance. Although intravitreal administration enables accumulation of high drug concentrations in the retina, the risk of drug precipitation due to the lipophilic nature of drugs, short vitreal half-life upon injection, and patient noncompliance deters this route of administration [126]. 42

Moreover, the chronic nature of uveitis requires multiple and frequent injections that carry the risk of vitreous hemorrhage, retinal detachment, and cataract progression.

Implants (e.g., Retisert-a fluocinolone acetonide intravitreal implant) overcome many of the disadvantages associated with intravitreal injections; however, the surgical procedure and risk of drug precipitation may result in undesirable effects [127, 128]. Recently, the

FDA approved intravitreal dexamethasone implant Ozurdex™ (administered via intravitreal injection) for treating uveitis, affecting the posterior segment. However, it is associated with many adverse effects such as increased intraocular pressure, conjunctival hemorrhage, conjunctival hyperemia, cataract, ocular hypertension, and vitreous detachment [129]. Hence, a non-invasive topical delivery would enhance patient compliance and minimize the side-effects associated with intraocular implants and intravitreal injections.

From a clinical and cost perspective, designing a delivery strategy that allows topical application of existing drug molecules would be a realistic alternative and would overcome the problems associated with posterior uveitis therapy. The small molecules used in uveitis therapy are lipophilic in nature with limited water solubility, thereby posing problems in formulating them as aqueous eye drops. Mixed nanomicelles are vesicular systems used for delivery of lipophilic substances [130]. Though nanomicelles have been known for three decades, their potential as vehicles for topical ocular drug delivery has been investigated only recently [10, 11]. A recent study has demonstrated that topical application of a mixed nanomicellar formulation of 0.1% dexamethasone, prepared by using D-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E 43

TPGS) and octoxynol-40, resulted in retinal drug concentration in rabbits. Nanomicelles have the potential to improve the solubility of drugs and increase the ocular bioavailability by overcoming diffusion barriers [109]. Velagaleti et al. [108] developed a nanomicellar formulation of vitamin E TPGS and octoxynol-40 for voclosporin, and ocular distribution studies following topical application in rabbits resulted in therapeutic concentrations of voclosporin in the retina and choroid. However, the mechanism by which nanomicelles deliver the drug to the retina following topical administration is not completely understood. To increase the retinal bioavailability of lipophilic drugs following topical application, two major issues need to be addressed: (i) increasing the solubility of drugs in aqueous eye drops, and (ii) increasing the permeability of drugs through the conjunctiva and sclera. The mixed nanomicellar technology could be used to enhance the solubility of drugs and increase the penetration across the conjunctival epithelium by temporarily altering the tight junctions.

We intend to develop and evaluate a mixed nanomicellar formulation using polyoxyethylated amphiphilic compounds (polyoxyl 40 stearate and polysorbate 80) that are approved by the US FDA for ocular use [131]. Polyoxyl 40 stearate and polysorbate

80 have been previously used in commercial ophthalmic products and are generally regarded as non-toxic and non-irritant materials [131]. To the best of our knowledge, mixed nanomicellar formulations of polyoxyl 40 stearate (P40S) and polysorbate 80

(P80) were not reported in the literature. The objective of the present study was to develop a clear, clinically acceptable, mixed nanomicellar eye drop formulation of 0.1% dexamethasone (DEXSOLV) for treating posterior uveitis. 44

3.3. Materials and Methods

3.3.3. Materials

Dexamethasone (Lot C137572) was procured from PCCA (Houston, TX). Polysorbate 80

(Lot 20589) was procured from Fisher Scientific (Pittsburgh, PA). Polyoxyl–40–stearate

(Lot 109K0160V) was procured from Sigma Aldrich (St. Louis, MO). Ethanol (Lot

B0522876) was supplied by ACROS (Fair Lawn, NJ). Benzalkonium chloride (Lot

Y52651G15) was supplied by Ruger (Irvington, NJ). Polyvinyl pyrrolidine-K-29/32

(PVP-K-29/32) (Lot 70804) and polyvinyl pyrrolidine-K-90 (PVP-K-90) (Lot 58-3) were procured from GAF (New York, NY). Tryptic soy broth (Soyabean Casein Digest medium-BactoTM, Lot 2030828) and Mueller-Hinton broth (Lot 3240477) were purchased from Fisher Scientific (Pittsburgh, PA). High Performance Liquid

Chromatography (HPLC) solvents, including acetonitrile (Lot 121151) and methanol (Lot

113904), were supplied by Fisher Scientific (Pittsburgh, PA). Distilled deionized water was used for the preparation of nanomicellar eye drop formulations.

3.3.2. Animals

New Zealand White (NZW) albino adult male rabbits, weighing between 2.0 and 2.5 kg, were obtained from Robinson Services Incorporated (Mocksville, NC). The animals were housed in accordance with the Association for Assessment and Accreditation of

Laboratory Animal Care International (AAALACI) and U.S. Department of Agriculture 45

(USDA). The animal protocol was reviewed and approved by the Institutional Animal

Care and Use Committee (IACUC) at The University of Toledo (Toledo, OH). Studies were performed as per the Association for Research in Vision and Ophthalmology

(ARVO) guidelines.

3.3.3. Preparation of 0.1% dexamethasone loaded mixed nanomicelles (DEXSOLV)

Mixed nanomicelles were prepared by the rotary evaporation method with an optimized composition of P40S/P80 = 7/3 by weight. In brief, dexamethasone (10 mg) and P40S

(0.42 g) were dissolved in 5 ml of ethanol. Ethanol was then evaporated under a vacuum at 50°C, leaving behind a thin film of dexamethasone and P40S. The thin film was rehydrated using 5 ml of water containing P80 (0.18 g). The rehydrated formulation was increased to 10 ml with the addition of phosphate buffered saline (2X) containing 2 mg of benzalkonium chloride. The final nanomicellar formulation was passed through a 0.22

μm sterile Nylon membrane filter (Millex® Syringe Filter, Sterile, 0.22 μm).

3.3.4. HPLC analysis of dexamethasone

HPLC (Waters Alliance e2695 separation module, Milford, MA), equipped with a 2998

PDA detector and reverse-phase C8 column (5 µm, 100A, Luna, Torrance, CA, USA), was used to identify dexamethasone in the nanomicellar formulations. The formulation was analyzed by an isocratic method with a mobile phase containing water and

acetonitrile (50:50) pumped at a flow rate of 1.0 ml/min. The absorbance of dexamethasone was measured at a wavelength of 242 nm.

3.3.5. Characterization of mixed nanomicelles

3.3.5.1. Critical micellar concentration (CMC) determination

The CMC was determined by measuring the surface tension of P80, P40S, and mixed nanomicelles at varying concentrations [132]. Dilutions of P80 ranging between 3.04 -

45.6 μM, P40S ranging between 5 - 80 μM, and mixed nanomicelles ranging between 5 -

60 μM, were prepared and analyzed for surface tension.

3.3.5.2. Effect of surfactant concentration on the solubility of dexamethasone

Mixed nanomicelles were prepared by varying the total concentration of P40S and P80 in the 1% - 6% w/v formulations as mentioned above. Dexamethasone (10 mg) and a predetermined quantity of P40S were dissolved in 5 ml of ethanol. Ethanol was then evaporated under a vacuum at 50°C, leaving behind a thin film of dexamethasone and

P40S. The thin film was rehydrated using 5 ml of water with a predetermined quantity of

P80 under continuous sonication for 30 minutes. The rehydrated formulation was increased to 10 ml with the addition of phosphate buffered saline (2X) containing 2 mg of benzalkonium chloride. The final nanomicellar formulation was passed through a 0.22

μm sterile Nylon membrane filter (Millex® Syringe Filter, Sterile, 0.22 μm) and the dexamethasone content was analyzed using HPLC. 47

3.3.5.3. Determination of pH

The pH of DEXSOLV was determined using an Accumet® excel XL 25 pH meter (Fisher

Scientific, Pittsburgh, PA). The pH meter was calibrated with standard buffer solutions of pH 4, 7, and 10 before each use.

3.3.5.4. Determination of particle size and zeta potential

The particle size and zeta potential of blank nanomicellar formulation and DEXSOLV were determined by dynamic light scattering (DLS) (NICOMP 380 ZLS, Particle Sizing

Systems, CA) technique. The formulation was taken in a borosilicate glass disposable culture tube and placed in a sample chamber maintained at 23°C. The particle size measurements were done at a scattering angle of 90° with an avalanche photodiode array detector. For the zeta potential measurement, the formulation was taken in a standard 1 cm square glass cuvette and placed in a sample chamber maintained at 23°C. The scattering angle was 14.1° with electric field strength of 15 V/cm. The particle size and zeta potential were determined after filtering the formulation through a 0.22 μm sterile

Nylon membrane filter (Millex® Syringe Filter, Sterile, 0.22 μm). The measurements were done in triplicate.

3.3.5.5. Transmission Electron Microscopy (TEM)

The morphology of DEXSOLV was studied using a TEM (HITACHI HD-2300 A, Ultra- thin Film Evaluation System, Hitachi High Technologies America, Pleasanton, CA). The

TEM sample was prepared by placing a small drop of mixed nanomicellar formulation on a holey carbon 400 mesh copper grid (Ted Pella, Redding, CA). Negative staining was done with a 2% phosphotungstic acid solution (Fisher Scientific, Pittsburgh, PA). The copper grid was air dried overnight. The sample was analyzed and images were captured using TEM.

3.3.5.6. Differential Scanning Calorimetry (DSC)

Physical state and thermal properties of dexamethasone, blank nanomicelles, and

DEXSOLV were determined using a Differential Scanning Calorimeter (DSC) (822e

Mettler Toledo) equipped with a TSO801RO sample robot and TS0800GCI gas flow system attached to a nitrogen gas cylinder. The samples (5-10 mg) were placed and sealed in aluminum crucibles using the Mettler MT 5 microbalance. DSC studies were performed at a heating rate of 10°C/min over a wide range (20 - 350°C). Star-e software

V8.10 was used to obtain the scans. Nitrogen gas was purged at a rate of 20 ml/min.

3.3.5.7. In vitro drug release study

In vitro release of dexamethasone from DEXSOLV was performed using a dialysis bag

(Fisherbrand® Dialysis tubing, MWCO: 12,000-14,000 Da). One milliliter of the nanomicellar formulation was placed in the dialysis bag and sealed. The dialysis bag was introduced into a vial containing 45 ml of isotonic phosphate buffer, pH 7.4, containing

0.025% (w/v) P80 to maintain sink conditions. The vials were placed in a shaker bath at

37±0.5°C and 60 oscillations/min. One milliliter of sample was withdrawn at predetermined time points and replaced with an equal volume of fresh buffer. The study was carried out in triplicate and the samples were analyzed using HPLC.

3.3.5.8. Clarity test

The clarity of mixed nanomicelles was measured in the presence of phosphate buffer and viscosity enhancers such as PVP-K-29-32 and PVP-K-90. Samples were prepared by mixing the nanomicellar formulation (2X) with an equal volume of phosphate buffer

(2X), PVP-K-29-32 (1.8% w/v) and PVP-K-90 (1.2% w/v) [133]. The clarity of the nanomicellar formulation was examined visually using a black and white background and by measuring the absorbance at 400 nm using an UV-visible spectrophotometer (Agilent

8453, UV-Vis Spectroscopy System). Distilled deionized water was used as a blank.

3.3.5.9. Stability

3.3.5.9.1. Dilution stability in artificial tears

Dilution stability of mixed nanomicelles in artificial tears was determined using a UV- visible spectrophotometer (Agilent 8453, UV-Vis Spectroscopy System) in the presence of PVP-K-29-32 (1.8% w/v) and PVP-K-90 (1.2% w/v) [133]. Each sample was further mixed with different brands of artificial tears such as Refresh Tears® (Lubricant Eye

Drops, Equate); Visine Tears® (Lubricant Eye Drops, Johnson & Johnson Healthcare

Products); Artificial Tears® (Lubricant Eye Drops, Prestige Brands, Inc.); and Gentle®

(Lubricant Eye Drops, Equate) in 1:1, 1:15, and 1:10 ratios. The measurements were taken under ambient conditions. The absorbance of each sample was recorded at 400 nm.

3.3.5.9.2. Formulation stability

DEXSOLV was transferred into glass vials and then stored at 4°C and 25°C for 6 months. At regular time intervals during the storage period, samples were examined for drug precipitation, changes in pH, changes in particle size, and drug content.

3.3.5.10. Filtration efficiency

DEXSOLV was filtered through 0.22 µm sterile membrane syringe filters made of polytetrafluoroethylene (PTFE), polyethersulfone (PES) and nylon membranes. The filtered formulations were analyzed for drug content using HPLC, and the values were

compared with the centrifuged formulation. DEXSOLV was centrifuged at 1500 rpm for

15 min.

3.3.5.11. Sterility test

Sterility testing was carried out for DEXSOLV sterilized via the filtration method.

Validation of the sterilization method was performed using direct and plate inoculation as per our published protocol [134]. Trypsin soy broth (TSB) was used for direct (tube) inoculation method. For the direct inoculation, a negative control vial, positive control vial, positive sample control vial, and aseptically filtered DEXSOLV vial were prepared.

The negative control vial contained 1 ml of sterile water and 9 ml of the uninoculated medium. Liquid culture of Staphylococcus aureus Rosenbach ATCC BAA 1692 was grown in TSB at 37°C for 24 h in a shaking water bath. The liquid culture was diluted to achieve a final concentration of 102 CFU/ml. The positive control vial contained 1 ml of water containing 102 CFU/ml and 9 ml of uninoculated medium. The positive sample control vial contained 1 ml of nanomicellar formulation containing 102 CFU/ml and 9 ml of uninoculated medium. The aseptically filtered DEXSOLV vial contained 1 ml of the

DEXSOLV that was aseptically passed through a 0.22 μm sterile nylon membrane filter

(Millex® Syringe Filter, Sterile, 0.22 μm) and 9 ml of the uninoculated medium. The vials were incubated at 37°C. For the plate inoculation, samples of 100 μl were withdrawn by the direct inoculation method from each of the vials on days 0, 7 and 14.

These samples were transferred and uniformly spread onto Mueller Hinton (MH) agar

plates. The study was performed under aseptic conditions in a laminar air flow hood. The plates were then incubated at 37°C for 12 h and observed for bacterial growth.

3.3.5.12. Ocular irritation study after single administration of the blank nanomicellar

formulation

The ocular irritation potential of the blank nanomicellar formulation was carried out in three male NZW rabbits at the NAMSA facility, Toledo, OH. Prior to administration, all test and control eyes were judged clinically normal for rabbits by gross examination with an auxiliary light source. To detect any pre-existing corneal injury, the eyes were treated with a fluorescein stain, flushed with 0.9% sodium chloride USP solution, and observed with ultraviolet light in a dark room. A 0.1 ml dose was administered into the lower conjunctival sac of one eye of each rabbit, and the lid was gently held loose for 1 second.

The other eye of each rabbit remained untreated and served as the comparative control.

The animals were returned to cages following treatment. At 24, 48, and 72 h after dosing, the test eye of each rabbit was examined with an auxiliary light source and appropriate magnification, compared to the untreated control eye, and graded for ocular irritation. To detect or confirm corneal injury, the test eye was treated with fluorescein stain, flushed with 0.9% sodium chloride USP solution, and examined in a dark room with an ultraviolet lamp at 24 hours after treatment. Reactions were scored in accordance with the

FHSA-modified Draize scoring criteria based on any alterations to the cornea (0-4), iris

(0-2), and conjunctiva for redness (0-3) and the eyelids for chemosis (0-4) [135].

3.3.5.13. Ocular tissue distribution of dexamethasone after single administration of

DEXSOLV

Ocular tissue distribution of dexamethasone after a single topical administration of the final nanomicellar formulation was carried out in five NZW rabbits. Eye drops (100 μL of final nanomicellar formulation/eye) were administered in the left eye of the rabbits.

Right eyes were used as a control. After 60 minutes, rabbits were euthanized using 1 ml of intravenous injection of euthanasia into the marginal ear vein. Eyeballs were then enucleated immediately within a few seconds and cleaned with cold phosphate buffer to remove any drug adsorbed onto the surface. Aqueous humor was withdrawn by limbal paracentesis and vitreous humor was aspirated using a 1 ml tuberculin syringe after making a tiny incision at the sclera–limbus junction. The eyeball was dissected and tissues such as the conjunctiva, cornea, iris-ciliary body, lens, retina-choroid, and sclera were collected, dried with Kim wipes®, and stored in pre-weighed vials. All tissue samples were stored at -80°C before further analysis. Ocular tissue concentrations of dexamethasone were analyzed by a validated LCMS/MS method described earlier from our lab [136, 137].

3.3.5.14. Statistical analysis

All the data presented are expressed as mean and standard deviation (mean± SD) with the number of data replicates in each study. The two-tailed Student’s t-test was used to evaluate the statistical differences, and a value of p < 0.05 was considered significant.

3.4. Results and Discussion

Nanomicelles of dexamethasone (DEXSOLV) were formulated with an optimized composition of P40S/P80 = 7/3 by weight. The nanomicelles of P40S and P80 could efficiently solubilize 0.1% w/v dexamethasone in their cores when the total surfactant concentration was 6% w/v concentration (Fig. 3-1).

A B

Dexamethasone

DEXSOLV Dexamethasone P80 eye drops, 0.1% P40S

FigFigure 1: 3 -(a)1: (A) DEXSOLV DEXSOLV eye drops, eye (B) drops, Structure of (b) a mixed Structure nanomicelle of a mixed nanomicelle.

Dexamethasone is poorly soluble in water and thus it was expected to demonstrate a good solubility when incorporated into mixed nanomicelles of P80 and P40S. Mixed nanomicelles prepared by varying the total concentration of P40S and P80 in the formulation in between 1% - 5% w/v resulted in a turbid solution, indicating the

incomplete solubility of dexamethasone. The concentration of dexamethasone in the solution increased with the concentration of P40S and P80. At a surfactant concentration of 6% w/v, the mixed nanomicellar formulation was able to dissolve 0.1% w/v of dexamethasone (Table 3.1). The CMC of surfactants, P80 and P40S, and the blank nanomicellar formulation was performed by measuring the change of surface tension.

The CMC values of P80 and P40S were found to be 12 µM and 40 µM, respectively

(Figs. 3-2A and 3-2B). The CMC value of the blank nanomicellar formulation was found to be 35 µM (Fig. 3-2C). The CMC value of the nanomicellar formulation was an intermediate value between individual CMC values of the surfactants [138].

Table 3.1: Solubility study of dexamethasone with increasing surfactant concentration

Amount of P40S and P80 (%w/v) Solubility of dexamethasone (%w/v)

1 0.030

2 0.054

3 0.065

4 0.085

5 0.098

6 0.100

60 ) 2 A 50 40 30 20 10

Surface Tension (g/s Tension Surface 0 0 10 20 30 40 50 P 80 Concentrations (μM)

B 40

Surface Tension (g/s2) Tension Surface 0 15 25 35 45 55 65 75 85 P40S Concentrations (µM)

C 50 40 30 20 10

Surface Tension (g/s2) Tension Surface 0 0 20 40 60 80 Blank Formulation Concentrations (µM)

Figure 3-2: Variation of the surface tension with surface concentration for (A) P80, (B)

P40S, and (C) Blank formulation

Nanomicelles prepared with a total surfactant concentration of 6% w/v concentration and

0.1% w/v dexamethasone (DEXSOLV) were characterized for particle size, pH, morphology, and drug content and in vitro release. The pH of DEXSOLV was found to be 7.2, which is close to the pH of normal tears. The pH of an ophthalmic preparation is important for proper preservation, since the stability of ophthalmic drugs will depend on the pH of their environment. The pH of an ophthalmic preparation would also influence the comfort, safety, and activity of the product [139]. DEXSOLV was dispersed in a 2%

(w/v) phosphotungstic acid negative stain, and the morphology was analyzed by TEM.

The TEM images revealed the spherical shape of mixed nanomicelles with a size ranging between 15-30 nm (Fig. 3-3). The size of nanomicelles from the TEM is close to the particle size values obtained using the DLS technique. No visible drug particles were observed in the TEM.

Figure 3-3: Transmission electron microscopy image of mixed nanomicelles

The sizes of mixed nanomicelles usually range between 10 to 100 nm [140]. The particle size determination was carried out for blank and drug-loaded mixed nanomicellar formulations. The particle size of blank mixed nanomicelles and drug-loaded mixed nanomicelles was small with a narrow distribution (Figs. 3-4A and 3-4C). The mean diameter and the polydispersity index (PDI) of blank and drug-loaded nanomicelles were

13.3 ± 0.4 nm and 14.5 ± 0.4 nm, respectively. The higher particle size of DEXSOLV as compared to blank nanomicelles could be attributed to the incorporation of dexamethasone (Mol. Wt.: 392.46 g/mol) within the core of nanomicelles [141]. The surface charge of mixed micelles can be determined by zeta potential values. The zeta potential of blank and drug-loaded mixed nanomicellar formulations was found to be neutral (Figs. 3-4B and 3-4D). The zeta potential of DEXSOLV was found to be 0.23 mV. The surface charge of nanomicelles depends on the nature of surfactants used in the preparation of nanomicelles. The nonionic nature of P40S and P80 tends to reduce the absolute zeta potential value.

DSC was performed to verify the absence of any un-dissolved dexamethasone in the mixed nanomicellar formulation (Fig. 3-5). DSC was carried out for dexamethasone, blank nanomicelles, and DEXSOLV. Pure dexamethasone exhibited a characteristic sharp endothermic peak with an onset at 243°C and a peak temperature of 262°C, which corresponds to its melting point [142]. Blank nanomicelles showed an endothermic peak with an onset at 90°C and a peak temperature of 100°C, which mostly corresponds to water [143]. DEXSOLV showed an endothermic peak with an onset at 97°C and a peak temperature of 101°C. The pure dexamethasone peak was absent in the DEXSOLV 59

thermogram, which indicated the absence of the crystalline form of dexamethasone. The complete disappearance of the melting point peak (243°C) in DEXSOLV represents the absence of the free form of the drug, which confirms the presence of dexamethasone molecules in the nanomicellar core.

Figure 3-4: Particle size and zeta potential curves of mixed nanomicellar formulation,

C (A and B) blank mixed nanomicellar formulation, and (C and D) drug-loaded mixed

nanomicellar formulation. 61

Figure 3-5: DSC thermogram of (A) Pure dexamethasone, (B) Blank nanomicelles, and

A HPLC method for the analysis of dexamethasone was successfully established and validated. The retention time was found to be 2.9 min. A stock solution of 1 mg/ml of dexamethasone was prepared in methanol. Calibration standards ranging from 1 - 50

μg/ml were prepared in the mobile phase (Fig. 3-6A). Each calibration standard was analyzed in triplicate, and the average peak area was plotted against the concentration.

The standard curve obtained was linear with an r2 value of 0.9975. The percentage recovery of dexamethasone ranged from 98.82-104.43%. The intra- and inter-assay precisions of dexamethasone were satisfactory; the relative standard deviations did not exceed 2%. The limit of detection and limit of quantification of dexamethasone were found to be 164.48 ng/ml and 498.4 ng/ml, respectively. A sample chromatogram of dexamethasone is shown in Fig. 3-6B.

1200000

1000000 y = 19523x + 18880

R² = 0.9975

800000

600000

400000 Peak Area (AU) Area Peak 200000

0 0 10 20 30 40 50 Dexamethasone concentration (ug/ml)

Figure 3-6: (A) Calibration curve of dexamethasone (B) Sample HPLC chromatogram of

dexamethasone 63

The in vitro release of dexamethasone from DEXSOLV was investigated using a dialysis membrane. The sink condition was maintained by the addition of 0.025% w/v P80 in the release medium. The nanomicelles showed a 100% release of dexamethasone in a sustained manner over a period of 8 h (Fig. 3-7). No visual change was observed in the nanomicellar formulation upon incubation with the release medium at 37°C, which indicated the stability of the drug-loaded nanomicelles in the presence of the release medium.

100

release 40

Cumulative percent dexamethasone dexamethasone percent Cumulative 0 0 2 4 6 8 10

Time (hours)

Figure 3-7: In vitro drug release of dexamethasone from mixed nanomicelles

The absorbance of DEXSOLV was found to be less than 0.1 at 400 nm (Table 3.2).

DEXSOLV was a clear solution without any precipitation of the drug. This indicated the stability of DEXSOLV in the buffer solution and different grades of PVP, which are commonly used as excipients in eye drops. The effect of dilution on the stability of nanomicelles was studied in the presence of artificial tear fluids. The formulation was diluted 2X, 10X, and 15X times with various marketed artificial tears. The mixture of

DEXSOLV drug-loaded micelles showed a clear solution without any precipitation in the presence of buffer and various grades of PVP (Table 3.3).

Table 3.2: Clarity Test of 0.1% dexamethasone mixed nanomicelles Table 2: Clarity test

Label and Ingredients Abs<400nm> Basic formulation (2X) + 0.0372

Buffer mixture (2X) + PVP-K-29-32 Basic formulation (2X) + 0.0387 Buffer mixture (2X) + PVP-K-90

Table 3.3: Artificial tear dilution test of 0.1% dexamethasone mixed nanomicelles Table 1: Artificial tear dilution test

Formulation Type of tear fluid Dilution Factor Abs<400nm> Refresh Tears 2x 3.07E-02 5x 1.16E-02 10x 3.21E-02 Visine tears 2X 2.71E-02 5X 2.79E-02 10X 2.57E-02 PVP-K-29-32 Artificial Tears 2X 3.87E-02 5 X 3.39E-02

10 X 8.31E-02 Gentle Tears 2X 2.39E-02 5 X 4.26E-02 10 X 6.09E-02 Refresh Tears 2x 8.03E-02 5x 4.48E-02 10x 4.24E-02

Visine tears 2X 3.29E-02 5X 6.71E-02 10X 3.05E-02 PVP-K-90 Artificial Tears 2X 3.54E-02 5 X 3.95E-02 10 X 4.11E-02 Gentle Tears 2X 2.49E-02

5 X 3.86E-02 10 X 3.53E-02

No increase in turbidity or precipitation of dexamethasone was observed. This indicates the stability of DEXSOLV in the tear dilution. DEXSOLV mixtures were stored at 4°C and 25°C for six months. At regular time intervals DEXSOLV was evaluated for changes in physical characteristics such as particle size, pH, and drug content. No changes were observed in the particle size, pH, and drug content for up to 6 months at 4°C and 25°C 66

(Table 3.4). The vials that were turbid at 4°C turned clear at room temperature. No visible change was observed during the six-month evaluation period in vials stored at 25°C.

Also, there was no precipitation of dexamethasone during the six-month period. This indicated the physical and chemical stability of DEXSOLV at 4°C and 25°C for at least 6 months.

Table 3.4: Formulation stability of DEXSOLV Table 4: Stability Study Temp. 0 Month 1 Month 2 Month 4 Month 6 Month Slightly Turbid, Slightly Turbid, Visual Clarity Clear Clear Clear Clear at room Clear at room temperature temperature 4°C Particle Size 12.9 ± 6.8 7.2 ± 2.9 11.4 ± 4.6 11.9 ± 5.4 10.9 ± 4.7 PDI 6.45 5.54 4.72 3.03 5.63 pH 5.57 5.45 5.54 5.43 5.42 Drug Content (%) 103.27 103.29 94.62 100.32 93.93 Visual Clarity Clear Clear Clear Clear Clear Particle Size 11.5 ± 4.6 7.4 ± 2.7 10.9 ± 3.7 10.8 ± 3.8 11.0 ± 3.9 25°C PDI 3.96 9.96 7.29 4.41 12.58 pH 5.33 4.96 4.63 3.88 3.75 Drug Content (%) 97.95 97.95 95.87 106.96 98.33

Filtration efficiency of DEXSOLV was studied with various types of sterile filter membranes. PTFE, PES and nylon membranes were found to be acceptable for sterilization. The drug content of the filtered nanomicellar formulation was compared with the centrifuged sample. The recovery of dexamethasone was found to be in the range of 95% - 100% in filtered samples (Table 3.5).

Table 3.5: Filtration efficiency test of 0.1% dexamethasone mixed nanomicellar

formulation. Table 5: Filtration Efficiency Test Sample Peak Area Recovery Drug (uV*sec) (ug/ml) Content (%) Polytetrafluoroethylene (PTFE) Membrane 1 203207 9.44 94.41 2 203552 9.46 94.59 3 204208 9.49 94.93 Polyethersulfone (PES) Membrane 1 210064 9.79 97.93 2 209545 9.77 97.66 3 209026 9.74 97.40 Nylon Membrane 1 209246 9.75 97.51 2 209245 9.75 97.51 3 208286 9.70 97.02 Centrifuged Sample 1 219864 10.29 102.95 2 216891 10.14 101.42 3 220304 10.32 103.17

Based on USP guidelines, the sterility of DEXSOLV was analyzed using direct inoculation and plate inoculation techniques in a TSB broth. The nanomicellar formulation was aseptically filtered through a 0.2 µm syringe filter. The vials were prepared and stored as per the method described. Except for the positive sample control vial, all other vials for the direct inoculation method were clear throughout the 14-day test period. The samples were withdrawn from the test tubes on days 0, 7, and 14 and were transferred onto MH agar plates for the plate inoculation method. The plates were examined for microbial growth after 24 h of incubation at 35°C, and the presence and absence of microbial growth is shown in Table 5.6. The positive control test tubes showed turbidity and microbial growth on the plates. Plates with negative control sample and the aseptically filtered formulation did not show any microbial growth throughout the

14-day test period. This indicated that the formulation can remain sterile for at least 14 days (Fig. 3-8).

Table 3.6: Sterility validation test performed on MH Agar plates indicating the presence

(+) or absence (-) of microbial growth on days 0, 7 and 14

Negative Positive Positive Aseptically Control Control Sample Control Filtered Formulation Day 0 - + + -

Day 7 - + + -

Day 14 - + + -

Figure 3-8: Sterility validation test performed on MH Agar plates indicating no bacterial

growth at 14 days 70

A preliminary ocular irritation study was carried out on three rabbits. One eye of each rabbit was treated with a blank mixed nanomicellar formulation. This study was performed to examine any alterations to the cornea, iris, conjunctiva, and eyelids. The rabbit eyes were examined and graded for ocular irritation after 24, 48, and 72 h (Table

3.7). The treated eyes were compared to the untreated eye to determine any changes. No irritation or redness was observed in the treated eyes as compared to the untreated control rabbit eyes.

Table 3.7: Ocular irritation study in rabbit eyes using a blank mixed nanomicellar

formulation, values are expressed as mean ± SD, n=3

Items Scored 24 h 48 h 72 h Results Cornea 0 0 0 ( - ) Iris 0 0 0 Redness* 0 0 0 Chemosis* 0 0 0 Fluorescien Exam ( - ) NA NA * = Conjunctival tissue, ( - ) = Negative NA = Not applicable

Following a single topical administration, tissue concentrations of dexamethasone (mean

± standard deviation, n = 5) in the cornea, iris-ciliary body, conjunctiva, aqueous humor, lens, sclera, and retina-choroid were found to be 293.44±81.43, 181.85±69.48,

139.51±89.40, 38.13±23.40, 6.91±2.48, 86.94±31.70, and 43.27±20.21 ng/gm of tissue, respectively (Fig. 3-9). The rank-order in terms of greatest to lowest concentration was cornea>iris-ciliary body>conjunctiva>sclera>retina-choroid>aqueous humor>lens.

Dexamethasone was able to penetrate into the retina-choroid due to its hydrophobic nature. The lipophilic nature of dexamethasone might have restricted its penetration into the vitreous humor, resulting in vitreal concentrations below the limit of quantification.

Figure 3-9: Dexamethasone concentration in ocular tissues following a single topical

administration of DEXSOLV, n=5

3.5. Conclusion

In conclusion, we demonstrated that amphiphilic molecules such as P40S and P80, when mixed in the proper ratios, form nanomicelles that can efficiently solubilize hydrophobic drugs such as dexamethasone in their core. At a surfactant concentration of 6% w/v, the mixed nanomicellar formulation was able to dissolve 0.1% w/v of dexamethasone. No irritation or redness was observed in the treated eyes as compared to the untreated control rabbit eyes following single and repeated topical administration. Ocular distribution studies following topical application of DEXSOLV in rabbits revealed therapeutic concentrations of dexamethasone in the retina and choroid. Using this eye drop formulation, dexamethasone concentrations could be maintained well above the minimum effective concentrations following topical administration. The proposed topical therapy would enhance patient compliance and minimize the side-effects associated with intraocular implants and intravitreal injections in posterior uveitis therapy.

Chapter 4 Preclinical Evaluation of a Nanomicellar Eye Drop Formulation of Dexamethasone for Posterior Uveitis Following Repeated Topical Administration

4.1. Abstract

Our previous study has shown that nanomicelles of polyoxyl 40 stearate (P40S) and polysorbate 80 (P80) could efficiently solubilize 0.1 % dexamethasone (DEXSOLV) in their cores resulting in a stable aqueous eye drop formulation. The objective of this study was to evaluate the ocular tolerance and tissue distribution of dexamethasone in New

Zealand White (NZW) albino adult male rabbits following repeated administration (2 times per day for 7 days) of DEXSOLV. No irritation or redness was observed in the treated eyes as compared to the untreated control rabbit eyes. Tissue concentrations of dexamethasone (mean ± standard deviation, n = 6) in the cornea, iris-ciliary body, conjunctiva, aqueous humor, lens, sclera, retina-choroid and vitreous humor were found to be 333.83±50.65, 172.15±24.57, 71.13±24.52, 55.13±11.70, 12.88±3.40,

112.75±53.09, 67.32±26.49, and 3.85±1.75 ng/gm of tissue, respectively. The findings of this investigation indicate that DEXSOLV might be clinically effective and could be used as a new treatment modality for delivering dexamethasone to the back of the eye tissues for treating posterior uveitis following topical administration.

4.2. Introduction

(toxoplasmosis) infections might play an important role [53]. Based on the structures affected, uveitis can be categorized into four forms: anterior, intermediate, posterior, and pan-uveitic forms. Anterior uveitis is the most common form of uveitis, affecting the iris and anterior chamber, while posterior uveitis affects primarily the retina and choroid.

Intermediate uveitis is characterized by inflammation in the vitreous cavity, while panuveitis affects all layers of uvea [54]. Posterior uveitis therapy requires localized delivery of drugs to the choroid layer, retina, and retinal vessels. Topical eye drop formulations fail to deliver the drugs to the retina as the anatomical and physiological properties that effectively protect the eye, hinder the efficient absorption of drugs [144].

Uveitis patients are generally treated using oral corticosteroids that often result in systemic side effects. Even the local administration of corticosteroids in the form of intravitreal injections and implants is associated with treatment-limiting side effects [126,

145]. The inherent potential side effects associated with intravitreal injections such as increased intraocular pressure, hemorrhage, retinal detachment, cataract, endophthalmitis, lead to complications limiting long term therapy [126]. Steroid administration to the posterior segment through implants is a promising strategy in the treatment of uveitis.

Fluocinolone acetonide implant (Retisert®, Bausch & Lomb) is available on the market

for treating posterior uveitis. Though implants overcome many of the disadvantages associated with intravitreal injections, surgical procedure and risk of drug precipitation due to poor solubility may cause side effects [60, 126]. Steroid sparing strategies such as immunosuppressant drugs (e.g., rapamycin) are also increasingly being used in order to avoid the complications of chronic steroid use, such as cataract and glaucoma. Despite the presence of a considerable number of new drugs, an effective delivery system for uveitis does not yet exist.

New treatment modalities to facilitate the topical delivery of drugs are under development. There are currently no marketed topical formulations for posterior segment diseases, with only a few compounds that are undergoing testing in clinical trials.

Velagaleti et al. [108] developed a nanomicellar formulation of vitamin E TPGS and octoxynol-40 for voclosporin and ocular distribution studies following topical application in rabbits resulted in therapeutic concentrations of voclosporin in the retina and choroid.

Nanomicelles are spherical in structure with size in the nanometer range. They are formed spontaneously when amphiphilic molecules consisting of hydrophobic and hydrophilic segments are dissolved in water [146, 147]. A mixed nanomicellar system is made up of two amphiphilic molecules and has superior stability and solubilization potential compared to conventional nanomicelles. Nanomicelles have the potential to improve the solubility of lipophilic drugs and increase the ocular bioavailability by overcoming diffusion barriers [109]. Though nanomicelles have been known for three decades, their potential as vehicles for topical ocular drug delivery has been investigated

recently [108, 148]. We have developed and characterized a mixed nanomicellar formulation of dexamethasone using polyoxyethylated amphiphilic compounds (polyoxyl

40 stearate and polysorbate 80) that are approved by US FDA for ocular use [131]. The objective of the present preclinical study was to evaluate the ocular tolerance and tissue distribution of dexamethasone in New Zealand White (NZW) albino adult male rabbits following multiple administration.

4.3. Materials and Methods

New Zealand White (NZW) albino adult male rabbits, weighed between 2.0 and 2.5 kg, were obtained from the Robinson Services Incorporated (Mocksville, NC). The animals were housed in accordance with the Association for Assessment and Accreditation of

Laboratory Animal Care International (AAALACI) and U.S. Department of Agriculture

(USDA). The animal protocol was reviewed and approved by the Institutional Animal

Care and Use Committee (IACUC) at The University of Toledo (Toledo, OH). Studies were performed as per the Association for Research in Vision and Ophthalmology

(ARVO) guidelines.

Eyes were examined for gross abnormalities using 2% ophthalmic fluorescein sodium, and healthy animals without pre-existing ocular irritation wereselected for the study. One hundred microlitiers of DEXSOLV was administered 2 times per day for 7 days in the left eye of the rabbits. Right eye was used as a control. Standard ocular examinations were performed 30 minutes after each dose and at 24, 48, and 72 hours after the last dose

and scored based on the modified Draize eye test [135]. Ocular changes were graded by a scoring system that includes rating any alterations to the cornea (0-4), iris (0-2) and conjunctiva for redness (0-3) and the eyelids for chemosis (0-4). Representative digital images were taken for test and control eyes daily. Euthanasia was performed 60 minutes after the last dose. Eyeballs were enucleated immediately and cleaned with an ice-cold phosphate buffer (pH 7.4) in order to remove any drug adsorbed onto the surface.

Aqueous humor was withdrawn by limbal paracentesis. The enucleated eyeballs were cut open and the following tissues were collected into preweighed vials: cornea, conjunctiva, iris-ciliary body, lens, aqueous humor, sclera, choroid-retina, and vitreous humor. All tissue samples were stored at -80°C until further analysis. Tissue samples were homogenized in 500 µL chilled (4°C) phosphate buffer (pH 7.4) for about 4 minutes with an Ultra-Turrax homogenizer (Ika T10 Basic, Staufen, Germany) in an ice bath.

Subsequently, 100 µL of the tissue homogenates (cornea, conjunctiva, iris-ciliary body, lens, sclera, choroid-retina, and vitreous humor) were collected for dexamethasone extraction. One hundred microliters of aqueous humor was used for extraction without further processing. Dexamethasone was extracted from ocular tissue homogenates by a simple liquid-liquid extraction, as per our published procedure, by liquid chromatography–tandem mass spectrometry (LC-MS/MS) [109, 137]. Triamcinolone acetonide (10.0 µg/ml) was used as internal standard. The extraction recovery of this method was found to be greater than 85% and this was calculated in ocular tissues as the ratio between the peak areas of extracted and unextracted samples. A Varian 320-MS

LC/MS triple quadruple mass spectrometer was used for analysis. High performance liquid chromatographic system consisted of Varian 212-LC Chromatography pump, Auto 78

sampler (Varian 469-LC, Varian Inc., Walnut Creek, CA) with a reversed phase Betasil

C18 column (50 × 4.6 mm i.d, 5µm, Thermo Scientific, Waltham, MA) attached to a guard column (Phenomenex, Torrance, CA). An isocratic mobile phase composed of acetonitrile and 0.1% formic acid in water (40:60) was pumped at a rate of 0.3 ml/min.

Dexamethasone and triamcinolone acetonide were detected with proton adducts at m/z

393.1 → 355.1 and 435.5 → 397.1, respectively, in multiple reaction monitoring (MRM) positive mode. The assay method was validated over a linear concentration range of 12.5-

250 ng/ml, with a correlation coefficient of 0.9988. The limits of detection and quantification of dexamethasone were found to be 0.78 ng/ml and 3.125 ng/ml, respectively. The intra- and interday precision (measured by the coefficient of variation,

CV%) was less than 3% and 5%, respectively. All the data presented are expressed as mean and standard deviation (mean± SD) with the number of data replicates in each study. Two-tailed Student’s t-test was used to evaluate the statistical differences and a value of p < 0.05 was considered significant.

4.4. Results and Discussion

No alterations were observed in the aforementioned ocular tissues in the presence of proper illumination. All animals were clinically normal throughout the study. Using an auxiliary light source, no significant corneal lesions, opacity, conjunctival chemosis, redness, discharge or iris alterations were observed in any of the rabbits (Table 4.1).

Table 4.1: Ocular irritation study in rabbit eyes following multiple drug loaded mixed

nanomicellar formulation, (mean ± SD; n=6), * = Conjunctiva

Items Days scored 1 2 3 4 5 6 7

Cornea 0 0 0 0 0 0 0

Iris 0 0 0 0 0 0 0

Redness* 0 0.063±0.25 0.063±0.25 0.25±0.45 0.188±0.40 0.063±0.25 0.063±0.25

Chemosis* 0 0 0 0 0 0 0

Representative digital images of the test and control eyes are shown in Figs. 4-1A and 4-

1B. Dexamethasone concentrations were observed in both anterior segment and posterior

segment tissues, including the retina-choroid. The tissue concentrations of

dexamethasone (mean ± standard deviation, n = 6) in the cornea, iris-ciliary body,

conjunctiva, aqueous humor, lens, sclera, retina-choroid and vitreous humor were found

to be 333.83±50.65, 172.15±24.57, 71.13±24.52, 55.13±11.70, 12.88±3.40,

112.75±53.09, 67.32±26.49, and 3.85±1.75 ng/gm of tissue, respectively (Fig. 4-2).

Figure 4-1: Rabbit eyes treated with (A) DEXSOLV, and (B) Control.

Figure 4-2: Dexamethasone concentration in ocular tissues following multiple

administration of DEXSOLV, n=6.

The rank-order from greatest to lowest concentration was cornea>iris-ciliary body>sclera>conjunctiva>retina-choroid>aqueous humor>lens>vitreous humor.

Dexamethasone concentration in vitreous humor was below the detection limit after single administration, however, upon repeated administration dexamethasone partitioned in to vitreous humor. In target tissues such as retina/choroid/vitreous humor, dexamethasone concentrations ranging from 10–4000 ng/gm are required for effective treatment of various inflammatory conditions [126]. Dexamethasone concentration in the choroid and retina was within the required concentration range.

4.5. Conclusion

In conclusion, we have demonstrated the delivery of dexamethasone to the back of eye tissues following topical application in New Zealand White (NZW) albino adult male rabbits. Using this eye drop formulation, dexamethasone concentrations could be maintained well above the minimum effective concentrations. This technology would profoundly benefit the patients who are currently on highly painful intravitreal injections and implants.

References

1. Gaudana, R., et al., Recent perspectives in ocular drug delivery. Pharm Res, 2009.

26(5): p. 1197-216.

2. Kaplan, H.J., Anatomy and function of the eye. Chem Immunol Allergy, 2007. 92:

p. 4-10.

3. Gehring, W.J. and K. Ikeo, < i> Pax 6: mastering eye morphogenesis and

eye evolution. Trends in genetics, 1999. 15(9): p. 371-377.

4. Land, M.F. and R.D. Fernald, The evolution of eyes. Annual review of

neuroscience, 1992. 15(1): p. 1-29.

5. Fernald, R.D., Evolving eyes. Int. J. Dev. Biol, 2004. 48: p. 701-705.

6. Martini, F., Human anatomy-/Frederic H. Martini, Michael J. Timmons, Robert

B. Tallitsch; with William C. Ober...[etc.]. 2005: San Francisco, PA [etc.]:

Pearson/Benjamin Cummings.

7. Dey, S. and A.K. Mitra, Transporters and receptors in ocular drug delivery:

opportunities and challenges. Expert opinion on drug delivery, 2005. 2(2): p. 201-

204.

8. Meek, K. and N.J. Fullwood, Corneal and scleral collagens—a microscopist’s

perspective. Micron, 2001. 32(3): p. 261-272.

9. Gray, H., Anatomy, descriptive and surgical. 1897: Lea Bros.

10. Civan, M.M. and A.D. Macknight, The ins and outs of aqueous humour secretion.

Experimental eye research, 2004. 78(3): p. 625-631.

11. Kinsey, V.E., The chemical composition and the osmotic pressure of the aqueous

humor and plasma of the rabbit. The Journal of general physiology, 1951. 34(3):

p. 389-402.

12. Goldmann, H., Minute volume of the aqueous in the anterior chamber of the

human eye in normal state and in primary glaucoma. Ophthalmologica. Journal

international d'ophtalmologie. International journal of ophthalmology. Zeitschrift

für Augenheilkunde, 1950. 120(1-2): p. 19.

13. Johnson, M. and K. Erickson, Mechanisms and routes of aqueous humor

drainage. Principles and Practice of Ophthalmology, 2000. 4: p. 2577-2595.

14. Brubaker, R., Flow of aqueous humor in humans [The Friedenwald Lecture].

Investigative ophthalmology & visual science, 1991. 32(13): p. 3145-3166.

15. Imesch, P.D., I.H. Wallow, and D.M. Albert, The color of the human eye: a

review of morphologic correlates and of some conditions that affect iridial

pigmentation. Survey of ophthalmology, 1997. 41: p. S117-S123.

16. Kaufman, P.L., L.A. Levin, and A. Alm, Adler's Physiology of the Eye. 2011:

Elsevier Health Sciences.

17. Tolentino, The Vitreous. Arch Ophthalmol, 1974. 92: p. 350-358.

18. Sebag, J., The Vitreous: Structure, Function, and Pathobiology. 1989. Springer-

Verlag, New York.

19. Summers Rada, J.A., S. Shelton, and T.T. Norton, The sclera and myopia.

Experimental eye research, 2006. 82(2): p. 185-200.

20. SANDBERG-LALL, M., et al., Type XIII collagen is widely expressed in the

adult and developing human eye and accentuated in the ciliary muscle, the optic

nerve and the neural retina. Experimental eye research, 2000. 70(4): p. 401-410.

21. Marshall, G.E., Human scleral elastic system: an immunoelectron microscopic

study. British journal of ophthalmology, 1995. 79(1): p. 57-64.

22. Thomas, O.E., Retina. In, S.J. Ryan, ed., 1989.

23. Jonas, J.B., et al., Human optic nerve fiber count and optic disc size. Investigative

ophthalmology & visual science, 1992. 33(6): p. 2012-2018.

24. Wade, N.J., Image, eye, and retina (invited review). J Opt Soc Am A Opt Image

Sci Vis, 2007. 24(5): p. 1229-49.

25. Knop, E. and N. Knop, Anatomy and immunology of the ocular surface. Chem

Immunol Allergy, 2007. 92: p. 36-49.

26. Newell, F.W., Ophthalmology, principles and concepts. 5th ed. 1982, St. Louis:

Mosby. x, 559 p.

27. Hawkins, B.S., et al., Epidemiology of age-related macular degeneration. Mol

Vis, 1999. 5: p. 26.

28. Leibowitz, H.M., et al., The Framingham Eye Study monograph: An

ophthalmological and epidemiological study of cataract, glaucoma, diabetic

retinopathy, macular degeneration, and visual acuity in a general population of

2631 adults, 1973-1975. Surv Ophthalmol, 1980. 24(Suppl): p. 335-610.

29. Iu, L.P. and A.K. Kwok, An update of treatment options for neovascular age-

related macular degeneration. Hong Kong Med J, 2007. 13(6): p. 460-70.

30. Sunness, J.S., et al., Visual function abnormalities and prognosis in eyes with age-

related geographic atrophy of the macula and good visual acuity.

Ophthalmology, 1997. 104(10): p. 1677-91.

31. Spilsbury, K., et al., Overexpression of vascular endothelial growth factor

(VEGF) in the retinal pigment epithelium leads to the development of choroidal

neovascularization. Am J Pathol, 2000. 157(1): p. 135-44.

32. Schmidt-Erfurth, U., et al., Histopathological changes following photodynamic

therapy in human eyes. Arch Ophthalmol, 2002. 120(6): p. 835-44.

33. Virgili, G. and A. Bini, Laser photocoagulation for neovascular age-related

macular degeneration. Cochrane Database Syst Rev, 2007(3): p. CD004763.

34. Costa, R.A., et al., Intravitreal bevacizumab for choroidal neovascularization

caused by AMD (IBeNA Study): results of a phase 1 dose-escalation study. Invest

Ophthalmol Vis Sci, 2006. 47(10): p. 4569-78.

35. Singh, S.R., et al., Intravenous transferrin, RGD peptide and dual-targeted

nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced

CNV. Gene Ther, 2009. 16(5): p. 645-59.

36. Gardner, T.W., et al., Diabetic retinopathy: more than meets the eye. Surv

Ophthalmol, 2002. 47 Suppl 2: p. S253-62.

37. Aiello, L.P., et al., Diabetic retinopathy. Diabetes Care, 1998. 21(1): p. 143-56.

38. Sanders, R.J. and M.R. Wilson, Diabetes-related eye disorders. J Natl Med

Assoc, 1993. 85(2): p. 104-8. 86

39. Mohamed, Q., M.C. Gillies, and T.Y. Wong, Management of diabetic

retinopathy: a systematic review. JAMA, 2007. 298(8): p. 902-16.

40. Yam, J.C. and A.K. Kwok, Update on the treatment of diabetic retinopathy. Hong

Kong Med J, 2007. 13(1): p. 46-60.

41. Park, K., et al., Nanoparticle-mediated expression of an angiogenic inhibitor

ameliorates ischemia-induced retinal neovascularization and diabetes-induced

retinal vascular leakage. Diabetes, 2009. 58(8): p. 1902-13.

42. Cao, Y., et al., Kringle 5 of plasminogen is a novel inhibitor of endothelial cell

growth. J Biol Chem, 1997. 272(36): p. 22924-8.

43. Klein, R., et al., The Wisconsin Epidemiologic Study of Diabetic Retinopathy

XXIII: the twenty-five-year incidence of macular edema in persons with type 1

diabetes. Ophthalmology, 2009. 116(3): p. 497-503.

44. Meyer, C.H., Current treatment approaches in diabetic macular edema.

Ophthalmologica, 2007. 221(2): p. 118-31.

45. Bandello, F., et al., Diabetic macular edema: classification, medical and laser

therapy. Semin Ophthalmol, 2003. 18(4): p. 251-8.

46. Aiello, L.P., et al., Vascular endothelial growth factor-induced retinal

permeability is mediated by protein kinase C in vivo and suppressed by an orally

effective beta-isoform-selective inhibitor. Diabetes, 1997. 46(9): p. 1473-80.

47. Nauck, M., et al., Induction of vascular endothelial growth factor by platelet-

activating factor and platelet-derived growth factor is downregulated by

corticosteroids. Am J Respir Cell Mol Biol, 1997. 16(4): p. 398-406.

48. Boddu, S.H., et al., Novel nanoparticulate gel formulations of steroids for the

treatment of macular edema. J Ocul Pharmacol Ther, 2010. 26(1): p. 37-48.

49. Lewis, H., T.M. Aaberg, and G.W. Abrams, Causes of failure after initial

vitreoretinal surgery for severe proliferative vitreoretinopathy. Am J Ophthalmol,

1991. 111(1): p. 8-14.

50. Ryan, S.J., The pathophysiology of proliferative vitreoretinopathy in its

management. Am J Ophthalmol, 1985. 100(1): p. 188-93.

51. Zhang, G., et al., Intraocular nanoparticle drug delivery: a pilot study using an

aerosol during pars plana vitrectomy. Invest Ophthalmol Vis Sci, 2007. 48(11):

p. 5243-9.

52. Asaria, R.H., et al., Adjuvant 5-fluorouracil and heparin prevents proliferative

vitreoretinopathy : Results from a randomized, double-blind, controlled clinical

trial. Ophthalmology, 2001. 108(7): p. 1179-83.

53. Forrester, J.V., Endogenous posterior uveitis. Br J Ophthalmol, 1990. 74(10): p.

620-3.

54. Forrester, J.V., Intermediate and posterior uveitis. Chem Immunol Allergy, 2007.

92: p. 228-43.

55. Lightman, S., Use of steroids and immunosuppressive drugs in the management of

posterior uveitis. Eye (Lond), 1991. 5 ( Pt 3): p. 294-8.

56. British Medical Association., Joint Formulary Committee (Great Britain), and

Pharmaceutical Society of Great Britain., British national formulary. The British

Medical Association and the Pharmaceutical Society of Great Britain: London. p.

v. 88

57. Sakai, T., et al., Therapeutic effect of stealth-type polymeric nanoparticles with

encapsulated betamethasone phosphate on experimental autoimmune

uveoretinitis. Invest Ophthalmol Vis Sci, 2011. 52(3): p. 1516-21.

58. Holbrook, J.T., et al., Visual loss in patients with cytomegalovirus retinitis and

acquired immunodeficiency syndrome before widespread availability of highly

active antiretroviral therapy. Arch Ophthalmol, 2003. 121(1): p. 99-107.

59. Jacobson, M.A., et al., Natural history and outcome of new AIDS-related

cytomegalovirus retinitis diagnosed in the era of highly active antiretroviral

therapy. Clin Infect Dis, 2000. 30(1): p. 231-3.

60. Duvvuri, S., S. Majumdar, and A.K. Mitra, Drug delivery to the retina:

challenges and opportunities. Expert Opin Biol Ther, 2003. 3(1): p. 45-56.

61. Sanborn, G.E., et al., Sustained-release ganciclovir therapy for treatment of

cytomegalovirus retinitis. Use of an intravitreal device. Arch Ophthalmol, 1992.

110(2): p. 188-95.

62. Hoyng, P.F. and L.M. van Beek, Pharmacological therapy for glaucoma: a

review. Drugs, 2000. 59(3): p. 411-34.

63. Allingham, R.R., Y. Liu, and D.J. Rhee, The genetics of primary open-angle

glaucoma: a review. Exp Eye Res, 2009. 88(4): p. 837-44.

64. Wildsoet, C., et al., Investigation of parameters influencing intraocular pressure

increases during sleep. Ophthalmic Physiol Opt, 1993. 13(4): p. 357-65.

65. Coleman, A.L. and L. Brigatti, The glaucomas. Minerva Med, 2001. 92(5): p.

365-79.

66. Sharts-Hopko, N.C. and C. Glynn-Milley, Primary open-angle glaucoma. Am J

Nurs, 2009. 109(2): p. 40-7; quiz 48.

67. Khaw, P.T., P. Shah, and A.R. Elkington, Glaucoma--1: diagnosis. BMJ, 2004.

328(7431): p. 97-9.

68. Rosenberg, L.F., Glaucoma: early detection and therapy for prevention of vision

loss. Am Fam Physician, 1995. 52(8): p. 2289-98, 2303-4.

69. Wheeler, L.A. and E. Woldemussie, Alpha-2 adrenergic receptor agonists are

neuroprotective in experimental models of glaucoma. Eur J Ophthalmol, 2001. 11

Suppl 2: p. S30-5.

70. Mediero, A., P. Alarma-Estrany, and J. Pintor, New treatments for ocular

hypertension. Auton Neurosci, 2009. 147(1-2): p. 14-9.

71. Bagnis, A., et al., Current and emerging medical therapies in the treatment of

glaucoma. Expert Opin Emerg Drugs. 16(2): p. 293-307.

72. Lavik, E., M.H. Kuehn, and Y.H. Kwon, Novel drug delivery systems for

glaucoma. Eye (Lond). 25(5): p. 578-86.

73. Doane, M., A. Jensen, and C. Dohlman, Penetration routes of topically applied

eye medications. American journal of ophthalmology, 1978. 85(3): p. 383-386.

74. Patton, T.F. and J.R. Robinson, Quantitative precorneal disposition of topically

applied pilocarpine nitrate in rabbit eyes. Journal of pharmaceutical sciences,

1976. 65(9): p. 1295-1301.

75. Anand, B.S., S. Dey, and A.K. Mitra, Current prodrug strategies via membrane

transporters/receptors. Expert opinion on biological therapy, 2002. 2(6): p. 607-

620. 90

76. Ranta, V.-P., et al., Barrier analysis of periocular drug delivery to the posterior

segment. Journal of Controlled Release, 2010. 148(1): p. 42-48.

77. Rhee, D.J., et al., Intraocular pressure alterations following intravitreal

triamcinolone acetonide. Br J Ophthalmol, 2006. 90(8): p. 999-1003.

78. Ozkiris, A. and K. Erkilic, Complications of intravitreal injection of

triamcinolone acetonide. Can J Ophthalmol, 2005. 40(1): p. 63-8.

79. Thompson, J.T., Cataract formation and other complications of intravitreal

triamcinolone for macular edema. Am J Ophthalmol, 2006. 141(4): p. 629-37.

80. Jonas, J.B., Intravitreal triamcinolone acetonide: a change in a paradigm.

Ophthalmic Res, 2006. 38(4): p. 218-45.

81. Janoria, K.G., et al., Novel approaches to retinal drug delivery. Expert Opin Drug

Deliv, 2007. 4(4): p. 371-88.

82. Castellarin, A. and D.J. Pieramici, Anterior segment complications following

periocular and intraocular injections. Ophthalmology clinics of North America,

2004. 17(4): p. 583-90, vii.

83. Maurice, D.M., Drug delivery to the posterior segment from drops. Surv

Ophthalmol, 2002. 47 Suppl 1: p. S41-52.

84. Acheampong, A.A., et al., Distribution of brimonidine into anterior and posterior

tissues of monkey, rabbit, and rat eyes. Drug Metab Dispos, 2002. 30(4): p. 421-9.

85. Koevary, S.B., J. Nussey, and S. Lake, Accumulation of topically applied porcine

insulin in the retina and optic nerve in normal and diabetic rats. Invest

Ophthalmol Vis Sci, 2002. 43(3): p. 797-804.

86. Kent, A.R., et al., Vitreous concentration of topically applied brimonidine tartrate

0.2%. Ophthalmology, 2001. 108(4): p. 784-7.

87. Osborne, N.N., et al., Topically applied betaxolol attenuates NMDA-induced

toxicity to ganglion cells and the effects of ischaemia to the retina. Exp Eye Res,

1999. 69(3): p. 331-42.

88. Acheampong, A.A., et al., Distribution of cyclosporin A in ocular tissues after

topical administration to albino rabbits and beagle dogs. Curr Eye Res, 1999.

18(2): p. 91-103.

89. Okamura, T., et al., Canine retinal arterial and arteriolar dilatation induced by

nipradilol, a possible glaucoma therapeutic. Pharmacology, 1996. 53(5): p. 302-

10.

90. Ahmed, I. and T.F. Patton, Importance of the noncorneal absorption route in

topical ophthalmic drug delivery. Invest Ophthalmol Vis Sci, 1985. 26(4): p. 584-

91. Ahmed, I. and T.F. Patton, Disposition of timolol and inulin in the rabbit eye

following corneal versus non-corneal absorption. International Journal of

Pharmaceutics, 1987. 38(1–3): p. 9-21.

92. Osborne, N.N., et al., Topical flunarizine reduces IOP and protects the retina

against ischemia-excitotoxicity. Invest Ophthalmol Vis Sci, 2002. 43(5): p. 1456-

64.

93. Kent, A.R., L. King, and L.R. Bartholomew, Vitreous concentration of topically

applied brimonidine-purite 0.15%. J Ocul Pharmacol Ther, 2006. 22(4): p. 242-6.

94. Mizuno, K., et al., Neuroprotective effect and intraocular penetration of

nipradilol, a beta-blocker with nitric oxide donative action. Invest Ophthalmol

Vis Sci, 2001. 42(3): p. 688-94.

95. Mizuno, K., et al., Route of penetration of topically instilled nipradilol into the

ipsilateral posterior retina. Invest Ophthalmol Vis Sci, 2009. 50(6): p. 2839-47.

96. Palanki, M.S., et al., Development of prodrug 4-chloro-3-(5-methyl-3-{[4-(2-

pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotria zin-7-yl)phenyl benzoate

(TG100801): a topically administered therapeutic candidate in clinical trials for

the treatment of age-related macular degeneration. J Med Chem, 2008. 51(6): p.

1546-59.

97. Tajika, T., A. Isowaki, and H. Sakaki, Ocular distribution of difluprednate

ophthalmic emulsion 0.05% in rabbits. J Ocul Pharmacol Ther, 2011. 27(1): p.

43-9.

98. Becker, U., et al., Tissue distribution of moxaverine-hydrochloride in the rabbit

eye and plasma. Journal of Ocular Pharmacology & Therapeutics, 2005. 21(3): p.

210-216.

99. Baklayan, G.A., et al., 24-Hour Evaluation of the Ocular Distribution of 14C-

Labeled Bromfenac Following Topical Instillation into the Eyes of New Zealand

White Rabbits. Journal of Ocular Pharmacology and Therapeutics, 2008. 24(4): p.

392-398.

100. Sigurdsson, H.H., et al., Topical and systemic absorption in delivery of

dexamethasone to the anterior and posterior segments of the eye. Acta

Ophthalmologica Scandinavica, 2007. 85(6): p. 598-602. 93

101. Loftsson, T., D. Hreinsdóttir, and E. Stefansson, Cyclodextrin microparticles for

drug delivery to the posterior segment of the eye: aqueous dexamethasone eye

drops. Journal of pharmacy and pharmacology, 2007. 59(5): p. 629-635.

102. Loftsson, T., et al., Dexamethasone delivery to posterior segment of the eye.

Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2007. 57(1-4): p.

585-589.

103. Martinez, A. and M. Sanchez-Salorio, A comparison of the long-term effects of

dorzolamide 2% and brinzolamide 1%, each added to timolol 0.5%, on

retrobulbar hemodynamics and intraocular pressure in open-angle glaucoma

patients. J Ocul Pharmacol Ther, 2009. 25(3): p. 239-48.

104. Kadam, R.S., et al., Ocular pharmacokinetics of dorzolamide and brinzolamide

after single and multiple topical dosing: implications for effects on ocular blood

flow. Drug Metabolism and Disposition, 2011. 39(9): p. 1529-1537.

105. Jansook, P., et al., Cyclodextrin solubilization of carbonic anhydrase inhibitor

drugs: formulation of dorzolamide eye drop microparticle suspension. European

Journal of Pharmaceutics and Biopharmaceutics, 2010. 76(2): p. 208-214.

106. Srirangam, R., et al., Evaluation of the intravenous and topical routes for ocular

delivery of hesperidin and hesperetin. J Ocul Pharmacol Ther, 2012. 28(6): p.

618-27.

107. Chen, Q., et al., The effect of deacetylated gellan gum on aesculin distribution in

the posterior segment of the eye after topical administration. Drug Delivery,

2012. 19(4): p. 194-201.

108. Velagaleti, P., et al., Topical Delivery of Hydrophobic Drugs Using a Novel

Mixed Nanomicellar Technology to Treat Diseases of the Anterior & Posterior

Segments of the Eye. Drug Delivery Technology, 2010 10(4): p. 42-47.

109. Earla, R., et al., Development and validation of a fast and sensitive bioanalytical

method for the quantitative determination of glucocorticoids--quantitative

measurement of dexamethasone in rabbit ocular matrices by liquid

chromatography tandem mass spectrometry. J Pharm Biomed Anal, 2010. 52(4):

p. 525-33.

110. Williams, K.A., et al., Topically applied antibody fragments penetrate into the

back of the rabbit eye. Eye (Lond), 2005. 19(8): p. 910-3.

111. Thiel, M.A., et al., Penetration of engineered antibody fragments into the eye.

Clinical & Experimental Immunology, 2002. 128(1): p. 67-74.

112. Furrer, E., et al., Pharmacokinetics and posterior segment biodistribution of

ESBA105, an anti–TNF-α single-chain antibody, upon topical administration to

the rabbit eye. Invest Ophthalmol Vis Sci, 2009. 50(2): p. 771-778.

113. Short, B.G., Safety evaluation of ocular drug delivery formulations: techniques

and practical considerations. Toxicologic pathology, 2008. 36(1): p. 49-62.

114. Chang-Lin, J.-E., et al., Pharmacokinetics and pharmacodynamics of a sustained-

release dexamethasone intravitreal implant. Investigative ophthalmology &

visual science, 2011. 52(1): p. 80-86.

115. Boddu, S., H. Gupta, and S. Patel, Drug Delivery to the Back of the Eye

Following Topical Administration: An Update on Research and Patenting

Activity. Recent patents on drug delivery & formulation, 2014. 95

116. Fialho, S., et al., Safety and pharmacokinetics of an intravitreal biodegradable

implant of dexamethasone acetate in rabbit eyes. Current eye research, 2006.

31(6): p. 525-534.

117. Brown, D.M., et al., Ranibizumab versus verteporfin for neovascular age-related

macular degeneration. N Engl J Med, 2006. 355(14): p. 1432-44.

118. Ip, M.S., et al., A randomized trial comparing the efficacy and safety of

intravitreal triamcinolone with observation to treat vision loss associated with

macular edema secondary to central retinal vein occlusion: the Standard Care vs

Corticosteroid for Retinal Vein Occlusion (SCORE) study report 5. Arch

Ophthalmol, 2009. 127(9): p. 1101-14.

119. Lindstrom, R. and T. Kim, Ocular permeation and inhibition of retinal

inflammation: an examination of data and expert opinion on the clinical utility of

nepafenac. Curr Med Res Opin, 2006. 22(2): p. 397-404.

120. Couch, S.M. and S.J. Bakri, Intravitreal triamcinolone for intraocular

inflammation and associated macular edema. Clinical ophthalmology (Auckland,

NZ), 2009. 3: p. 41.

121. Gritz, D.C. and I.G. Wong, Incidence and prevalence of uveitis in Northern

California; the Northern California Epidemiology of Uveitis Study.

Ophthalmology, 2004. 111(3): p. 491-500; discussion 500.

122. de Boer, J., N. Wulffraat, and A. Rothova, Visual loss in uveitis of childhood. Br J

Ophthalmol, 2003. 87(7): p. 879-84.

123. Suhler, E.B., et al., Incidence and prevalence of uveitis in Veterans Affairs

Medical Centers of the Pacific Northwest. Am J Ophthalmol, 2008. 146(6): p.

890-6 e8.

124. Janoria, K.G., et al., Novel approaches to retinal drug delivery. 2007.

125. Sai HS, B., Polymeric Nanoparticles for Ophthalmic Drug Delivery: An Update

on Research and Patenting Activity. Recent Patents on Nanomedicine, 2012. 2(2):

p. 96-112.

126. Boddu, S.H., et al., Novel nanoparticulate gel formulations of steroids for the

treatment of macular edema. Journal of Ocular Pharmacology and Therapeutics,

2010. 26(1): p. 37-48.

127. Arcinue, C.A., O.M. Cerón, and C.S. Foster, A comparison between the

fluocinolone acetonide (Retisert) and dexamethasone (Ozurdex) intravitreal

implants in uveitis. Journal of Ocular Pharmacology and Therapeutics, 2013.

29(5): p. 501-507.

128. Kompella, U.B. and H.F. Edelhauser, Drug Product Development for the Back of

the Eye. Vol. 2. 2011: Springer.

129. Haller, J.A., et al., Evaluation of the safety and performance of an applicator for a

novel intravitreal dexamethasone drug delivery system for the treatment of

macular edema. Retina, 2009. 29(1): p. 46-51.

130. Trivedi, R. and U.B. Kompella, Nanomicellar formulations for sustained drug

delivery: strategies and underlying principles. Nanomedicine (Lond). 5(3): p.

485-505.

131. Jiao, J., Polyoxyethylated nonionic surfactants and their applications in topical

ocular drug delivery. Adv Drug Deliv Rev, 2008. 60(15): p. 1663-73.

132. Bondarenko, A.I., R. Malli, and W.F. Graier, The GPR55 agonist

lysophosphatidylinositol directly activates intermediate-conductance Ca2+-

activated K+ channels. Pflügers Archiv-European Journal of Physiology, 2011.

462(2): p. 245-255.

133. Mitra, A.K., P.R. Velagaleti, and S. Natesan, Ophthalmic Compositions

Comprising Calcineurin Inhibitors or mTOR Inhibitors. 2011, Google Patents.

134. Nesamony, J., et al., Development and Characterization of Nanostructured Mists

with Potential for Actively Targeting Poorly Water-Soluble Compounds into the

Lungs. Pharmaceutical research, 2013. 30(10): p. 2625-2639.

135. Wilhelmus, K.R., The Draize eye test. Survey of ophthalmology, 2001. 45(6): p.

493-515.

136. Earla, R., et al., Development and validation of a fast and sensitive bioanalytical

method for the quantitative determination of glucocorticoids-Quantitative

measurement of dexamethasone in rabbit ocular matrices by liquid

chromatography tandem mass spectrometry. J Pharm Biomed Anal, 2010. 52(4):

p. 525-33.

137. Boddu, S.H., H. Gupta, and S.P. Bonam, Preclinical evaluation of a ricinoleic

acid poloxamer gel system for transdermal eyelid delivery. International journal

of pharmaceutics, 2014. 470(1): p. 158-161.

138. Li, L. and Y.B. Tan, Preparation and properties of mixed micelles made of

Pluronic polymer and PEG-PE. Journal of colloid and interface science, 2008.

317(1): p. 326-331.

139. Aldricha, D.S., et al., Ophthalmic Preparations.

140. Gan, L., et al., Self-assembled liquid crystalline nanoparticles as a novel

ophthalmic delivery system for dexamethasone: improving preocular retention

and ocular bioavailability. International journal of pharmaceutics, 2010. 396(1):

p. 179-187.

141. Song, H., et al., Development of Polysorbate 80/Phospholipid mixed micellar

formation for docetaxel and assessment of its in vivo distribution in animal

models. Nanoscale Research Letters, 2011. 6(1): p. 1-12.

142. Gómez-Gaete, C., et al., Encapsulation of dexamethasone into biodegradable

polymeric nanoparticles. International journal of pharmaceutics, 2007. 331(2): p.

153-159.

143. Girdthep, S., et al. Effect of Tween 80 on the Mechanical and Thermal Properties

of Solution-Cast Blends of Poly (lactic acid) and Cellulose Acetate Butyrate

Films. in Proceedings of International Conference on Chemistry and Chemical

Process (ICCCP 2011). 2011.

144. HS Boddu, S., Polymeric Nanoparticles for Ophthalmic Drug Delivery: An

Update on Research and Patenting Activity. Recent Patents on Nanomedicine,

2012. 2(2): p. 96-112.

145. Lowder, C., et al., Dexamethasone intravitreal implant for noninfectious

intermediate or posterior uveitis. Archives of ophthalmology, 2011. 129(5): p.

545-553.

146. Torchilin, V.P., Micellar nanocarriers: pharmaceutical perspectives. Pharm Res,

2007. 24(1): p. 1-16.

147. Aliabadi, H.M. and A. Lavasanifar, Polymeric micelles for drug delivery. Expert

Opin Drug Deliv, 2006. 3(1): p. 139-62.

148. Pepic, I., et al., A nonionic surfactant/chitosan micelle system in an innovative eye

drop formulation. J Pharm Sci, 2010. 99(10): p. 4317-25.

100