A Novel Protein Drug, Suprachoroidal Delivery, and Protein Sustained Release Systems for Choroidal Neovascularization

A NOVEL PROTEIN DRUG, SUPRACHOROIDAL DELIVERY, AND PROTEIN

SUSTAINED RELEASE SYSTEMS FOR CHOROIDAL NEOVASCULARIZATION

PUNEET TYAGI

M. Pharm., Hamdard University, India 2003

B. Pharm, CCS University, India 1997

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Pharmaceutical Sciences Program

2013

This thesis for Doctor of Philosophy degree by

Puneet Tyagi

has been approved for the

Pharmaceutical Sciences Program

Robert I. Scheinman, Chair

Uday B. Kompella, Advisor

Krishna Mallela

Ravi Mahalingam

Jeffrey Olson

Date: 12/05/13

Tyagi, Puneet. (Ph.D., Pharmaceutical Sciences)

A Novel Protein Drug, Suprachoroidal Delivery, and Protein Sustained Release Systems for Choroidal Neovascularization

Thesis directed by Professor Uday B. Kompella.

ABSTRACT

Ocular posterior segment diseases such as age related macular degeneration are leading causes of blindness worldwide. Identification of various new disease targets have led to the development of inhibitors of vascular endothelial growth factor such as bevacizumab, ranibizumab, and aflibercept. A critical barrier for the translation of any new promising drug intended for the posterior segment diseases is drug delivery to the back of the eye. Topical eye drop, which is the most convenient dosage form, typically does not achieve therapeutically effective drug levels in the retina. Systemic modes of administration are associated with high drug exposure to other organs, and hence, systemic toxicity. Frequent intravitreal injections can lead to retinal detachment and endophthalmitis.

The objective of this research was to create a novel transferrin-tumstatin fusion protein based on an endogenous antiangiogenic tumstatin and evaluate its efficacy in vitro and in vivo and compare it to bevacizumab and tumstatin. Following design, expression, and efficacy assessment, pharmacokinetics and safety of transferrin-tumstatin was assessed in rabbits. Suprachoroidal route of delivery was validated in rats and the delivery of sodium fluorescein by this route was compared to subconjunctival and

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intravitreal routes. A delivery system for sustained in vivo delivery of bevacizumab following suprachoroidal administration was developed.

Transferrin-tumstatin was superior to bevacizumab and tumstatin in inhibiting choroid endothelial cell proliferation, tube formation, and migration. The in vivo efficacy of transferrin-tumstatin was superior to bevacizumab and tumstatin in inhibiting CNV in a rat model. Transferrin-tumstatin was eliminated from the vitreous humor with a half life of about 2 days and was safe following intravitreal injection in rabbits. Further, suprachoroidal injections are feasible in rats and the extent and rate of delivery of sodium fluorescein to choroid-retina was the highest following suprachoroidal delivery, when compared to intravitreal and posterior subconjunctival injections. A light-activated in situ forming gel sustained the delivery of bevacizumab after suprachoroidal dosing.

The form and content of this abstract are approved. I recommend its publication.

Approved: Uday B. Kompella

This thesis is dedicated to my wife and son for their constant support and encouragement.

ACKNOWLEDGEMENTS

First and foremost, I would like to express my deepest regards and gratitude to Dr.

Uday B. Kompella for providing me the opportunity and support to work under his supervision. I am really thankful to him for always assisting me to his fullest extent under all circumstances and for his immense generosity and expertise as a teacher and for his patience and support over the years that I have spent in his laboratory. Under his guidance not only I could advance myself scientifically, I could also improve my writing and oral presentation skills by a tremendous amount. This thesis was possible only because of valuable insights provided by him as an accomplished scientist and advisor. I will forever cherish his guidance and look forward to his advice.

I am also grateful to other members of my thesis committee members – Dr.

Robert Scheinman, Dr. Krishna Mallela, Dr. Ravi Mahalingam, and Dr. Jeffrey Olson for their extremely valuable recommendations, suggestions, encouragement and insightful comments. It has been a privilege for me to have committee members as scientific guides.

I am forever grateful to them.

I am also thankful to all my past and present colleagues and friends in the

Department of Pharmaceutical Sciences – Ruchit Trivedi, Shelley Durazo, Shreya

Kulkarni, Sunil Vooturi, Arun Upadhyay, Rajendra Kadam, Swita R. Singh, Rinku Baid,

Jiban Jyoti, Jiban Anand, Vidhya Rao, Gajanan Jadhav, Ashish Thakur, Namdev Shelke,

Chandrasekar Durairaj, and Sneha Sundaram, for their joyous company and insightful comments. I am especially thankful to Ruchit Trivedi, Rajendra Kadam, and Swita Singh for being such wonderful friends and for being there at times of need.

I would also like to acknowledge the financial support from school of Pharmacy and from National Institute of Health.

Finally, I would like to acknowledge my parents for always believing me in all my decisions in life including the pursuit of this program. They are my greatest source of inspiration and strength. Last but not least I would like to thank my wife and son for their support, patience, understanding, and immense faith on me.

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TABLE OF CONTENTS

CHAPTER

I. STATEMENT OF PROBLEM ...... 1

Introduction ...... 1

Specific aims for the thesis ...... 8

II. INTRODUCTION ...... 11

Age related macular degeneration ...... 11

Basic definition of AMD ...... 12

Choroidal neovascularization ...... 14

Role of pro-angiogenic factors in CNV ...... 15

Therapeutic strategies for CNV ...... 17

Drawbacks of current therapeutic strategies of CNV ...... 19

Endogenous inhibitors of CNV ...... 21

Inhibition of angiogenesis by tumstatin ...... 23

Transferrin-tumstatin, a novel fusion protein for CNV ...... 24

Current techniques for drug delivery to the posterior region of the eye ...... 25

Suprachoroidal delivery for posterior region of the eye ...... 27

Sustained delivery systems for posterior region of the eye ...... 29

Sustained release systems for proteins ...... 31

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III. A NOVEL TUMSTATIN FUSION PROTEIN FOR CHOROIDAL NEOVASCULARIZATION ...... 33

Abstract ...... 33

Introduction ...... 34

Materials and methods ...... 37

Results ...... 45

Discussion ...... 56

Conclusions ...... 60

IV. PHARMACOKINETICS AND SAFETY OF A NOVEL TRANSFERRIN-TUMSTATIN FUSION PROTEIN IN RABBITS ...... 61

Abstract ...... 61

Introduction ...... 62

Materials and methods ...... 64

Results ...... 68

Discussion ...... 75

Conclusions ...... 76

V. COMPARISON OF SUPRACHOROIDAL DRUG DELIVERY WITH SUBCONJUNCTIVAL AND INTRAVITREAL ROUTES USING NONINVASIVE FLUOROPHOTOMETRY ...... 77

Abstract ...... 77

Introduction ...... 78

Materials and methods ...... 82

Results ...... 85

Discussion ...... 95

Conclusions ...... 101

VI. LIGHT ACTIVATED, IN SITU FORMING GEL FOR SUSTAINED SUPRACHOROIDAL DELIVERY OF BEVACIZUMAB ...... 102

Abstract ...... 102

Introduction ...... 104

Materials and methods ...... 107

Results ...... 113

Discussion ...... 126

Conclusions ...... 130

VII. SUMMARY AND FUTURE DIRECTIONS ...... 131

Specific Aim 1 ...... 135

Specific Aim 2 ...... 138

Specific Aim 3 ...... 140

Specific Aim 4 ...... 141

REFERENCES ...... 144

LIST OF TABLES

TABLE

2.1 FDA approved sustained delivery systems for delivery to the posterior region of the eye ...... 29

3.1 IC 50 values of tumstatin, Tf-T and bevacizumab in the three in vitro assays ...... 57

4.1 Clinical examination performed on New Zealand white rabbits before intravitreal injection, and 1 and 7 days after injection of Tf-T ...... 74

4.2 Comprehensive metabolic evaluation performed on New Zealand white rabbits before intravitreal injection, and at the end of 7 days after injection of Tf-T ...... 74

LIST OF FIGURES

FIGURE

2.1 Different stages of AMD ...... 13

2.2 Schematic representation of angiogenesis in CNV ...... 16

2.3 Schematic representation of intravitreal and suprachoroidal route of injection in the eye ...... 28

3.1 Characterization of Tf-T and tumstatin protein ...... 46

3.2 Anti-proliferative activity of Tf-T when compared to tumstatin and bevacizumab in RF/6A cells ...... 47

3.3 Inhibition of tube formation by Tf-T when compared to tumstatin and bevacizumab observed in RF/6A cells ...... 48

3.4 Inhibition of endothelial cell invasion by Tf-T when compared to tumstatin and bevacizumab observed in RF/6A cells ...... 49

3.5 Induction of endothelial cell apoptosis by Tf-T when compared to tumstatin and bevacizumab observed in RF/6A cells ...... 50

3.6 Basolateral secretion of Tf-T protein in RPE cells grown on Transwell TM filters ...... 52

3.7 In silico modeling of tumstatin and Tf-T ...... 53

3.8 Representative figure of internalization of Tf-T and tumstatin protein in RF/6A cells ...... 53

3.9 In vivo assessment of CNV lesion size in BN rats ...... 55

4.1 SDS PAGE gel of transferrin-tumstatin purified and used in the pharmacokinetic and safety study ...... 68

4.2 Intravitreal levels of alexa conjugated Tf-T equivalent to sodium fluorescein measured by ocular fluorophotometry ...... 69

4.3 Representative figure of rabbit eyes showing the appearance and disappearance of hyperemia ...... 70

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4.4 Fundus images showing vitreal floater observed in 1 out of 4 New Zealand white rabbits following intravitreal administration of Tf-T ...... 70

4.5 Intraocular pressure in rabbit eyes dosed with Tf-T. Contralateral eye is the undosed eye of the rabbits and serve as a control ...... 71

4.6 Angiography and bright field images rabbit eyes dosed with Tf-T ...... 72

5.1 Suprachoroidal injection of India ink between sclera and choroid-retina in SD rats ...... 87

5.2 Representative fluorophotometry scans attained using Fluorotron Master™ in Sprague Dawley rat eye ...... 88

5.3 Sodium fluorescein concentrations in choroid-retina, vitreous, and anterior chamber regions after injection in the suprachoroidal ...... 90

5.4 Sodium fluorescein concentration in choroid-retina, vitreous, and anterior chamber regions after posterior subconjunctival injection ...... 91

5.5 Sodium fluorescein concentrations in choroid-retina, vitreous, and anterior chamber regions after intravitreal injection ...... 92

5.6 Pharmacokinetic parameters estimated for sodium fluorescein after injection by suprachoroidal, intravitreal, and posterior subconjunctival routes in SD rats ...... 94

6.1 NMR spectra for (A) polycaprolactone diol, (B) isocyanoethyl methacrylate, and (C) polycaprolactone dimethacrylate ...... 114

6.2 Reaction scheme for the preparation of cross-linked gel ...... 115

6.3 Cumulative release of bevacizumab from the gel at three different cross-linking durations ...... 117

6.4 Size exclusion chromatograms of bevacizumab released from the gel prepared by 10 min cross-linking ...... 118

6.5 Circular dichroism spectra for of bevacizumab released from the gel prepared by 10 min cross-linking ...... 119

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6.6 Injection and in situ cross-linking of gel-forming polymers mixed with Alexa Fluor 488 dye conjugated bevacizumab in excised rabbit eyes ...... 120

6.7 Alexa Fluor 488 dye conjugated bevacizumab retained in the suprachoroidal space of SD rats ...... 122

6.8 In vitro toxicity of in situ forming gel in ARPE cells ...... 124

6.9 In vivo toxicity of in situ forming gel in SD rats ...... 125

6.10 Schematic representation of sustained delivery of bevacizumab from in situ forming gels ...... 126

7.1 Hypothetical mechanism of superior efficacy of transferrin-tumstatin over tumstatin ...... 137

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CHAPTER I

STATEMENT OF PROBLEM

The overall goal of this research was to develop a novel protein drug and sustained release delivery systems for protein drugs in order to improve the management of choroidal neovascularization in wet age related macular degeneration (AMD).

Introduction

Choroidal neovascularization (CNV) is defined as abnormal growth of blood vessels, originating in the choroid, which pass through Bruch's membrane and then proliferate into the retina (Green 1999). Factors including release of cytokines such as vascular endothelial growth factor (VEGF), inflammation, and oxidative stress may be the underlying causes of CNV. Although VEGF is currently thought to be the main culprit in CNV, growth factors such as pigment epithelium derived growth factor

(PEDF), fibroblast growth factor (FGF2), angiopoietins, and matrix metalloproteinases have also been implicated in the development of CNV (Das and McGuire 2003).

Since VEGF plays a central role in the development of CNV, therapies that target

VEGF are the obvious current treatment modalities. Ranibizumab (Lucentis TM ) and aflibercept (Eylea TM ), anti-VEGF protein therapeutics, have been shown in clinical trials to prevent the progression of CNV (Heier, Brown et al. 2013). Although VEGF inhibitory modalities are clinically successful, it is generally debated that inhibiting VEGF is not the optimal way to manage CNV development (Singh, Amin et al. 2005). Further, extracellularly acting VEGF inhibitory modalities fail to induce regression of the disease

(Singh, Amin et al. 2005) and improve vision in 60% of the patients suffering from the disease. Thus, new therapies and cellular targets are needed that not only prevent growth of CNV, but also regress the disease. To address these unmet needs, we aimed at creating a fusion protein based on tumstatin, an endogenous angiogenesis inhibitor, to treat CNV.

Tumstatin is derived from the C terminus non-collagenous domain (NC1) of collagen IV present in the basement membrane (Hamano, Zeisberg et al. 2003). Tumstatin plays a pivotal role in inhibiting neovascularization by binding to αVβ3 integrins, hindering the proliferation of endothelial cells and also by inducing endothelial cell apoptosis

(Sudhakar, Sugimoto et al. 2003).

Specifically, we aimed at the design of transferrin-tumstatin, which is expected to have many advantages over the current approved anti-VEGF agents. Transferrin receptor- mediated uptake and transcytosis (Qian, Li et al. 2002) can be utilized for enhancing delivery of proteins across cellular barriers, including retinal barriers. Further, transferrin receptor is expressed more in proliferating cells (Weller, Wiedemann et al. 1989) than quiescent cells, thereby allowing preferential delivery to proliferating cells. The other part of our fusion protein, tumstatin also has a cell specific delivery, with the tumstatin receptor, αVβ3 integrin only being found on activated endothelial cells and not on normal blood vessels(Friedlander, Theesfeld et al. 1996). Thus, tumstatin is expected to provide a path to target activated endothelial cells, without affecting physiological processes such as wound healing. Tumstatin has been reported to cause apoptosis in endothelial cells, and apoptosis caused by tumstatin can cause regression of proliferating blood vessels

(Maeshima, Colorado et al. 2000) and it might be advantageous in restoring normal vision. We hypothesize that transferrin- tumstatin, as a therapeutic agent, can potentially

treat CNV better than the standard of care and prevent vision loss in humans in the long run. Thus, the first aim of this study was to design, express, purify, characterize, and evaluate the efficacy of transferrin-tumstatin in vitro and in vivo in a disease model.

In order to develop transferrin-tumstatin for CNV, it is important to know its pharmacokinetics and safety after intravitreal injection, a clinically validated route of administration for protein drugs intended for the back of the eye. These studies are useful in advancing the drug molecule for future human use. Non-rodents such as rabbits are usually selected as the species of choice in ocular pharmacokinetic studies as the eyes are larger and comparable to human eye dimensions. Although rodents can be used for investigational purposes, rabbit model is particularly desirable for IND enabling preclinical pharmacokinetic studies. Pharmacokinetics can be monitored using discrete sampling or non-invasively using ocular fluorophotometry. Fluorophotometry is a method for tracing a drug in live animals and has been found to be safe and noninvasive to the animal. With this approach pharmacokinetics of a drug injected into the vitreous can be determined without anaesthetizing the animal. Traditionally, the procedure for assessing ocular pharmacokinetics includes sacrificing multiple animals at each time point followed by eye enucleation. Once the eye is enucleated, isolation of ocular tissues is performed, which is a tiresome and time consuming task. During the isolation of different tissues drug can migrate and subsequently affect the outcome. Compared to the traditional methods, in non-invasive ocular fluorophotometry technique, the same animal can be used to evaluate drug levels in different ocular tissues over multiple time points.

This allows a time course evaluation without the hassles associated with traditional methods of ocular pharmacokinetic studies. Ghate el al.(Ghate, Brooks et al. 2007) used

fluorophotometry to evaluate sodium fluorescein (NaF) pharmacokinetics in rabbit eyes in vivo and Lee et al.(Lee, Kim et al. 2008) used fluorophotometry to study transcleral delivery of Oregon green 488 conjugated triamcinolone acetonide in rabbit eyes.

Since vision is arguably the most important sense organ, conducting ocular a toxicity and tissue distribution study in animals is essential to ensure the safety of therapeutics injected into the eye prior to human administration. In order to assess the risk that transferrin-tumstatin poses to the eye, we also aimed at assessment of ocular safety in rabbits. Thus, the second aim of this study was to determine the pharmacokinetics and safety of transferrin-tumstatin in rabbits following a single intravitreal injection.

Delivery of therapeutics to the eye is most commonly achieved by topical instillation in the form of eye drops. Topical administration, although efficient in delivering drugs to the anterior segment of the eye, fails to deliver drugs to the posterior ocular tissues such as retina. As delivery to the back of the eye is negligible with topical application, intravitreal injections were developed and are gaining popularity. However, despite the growing use, intravitreal injections are highly invasive in nature and cataract, retinal detachment, vitreous hemorrhage, and endophthalmitis are associated with the intravitreal route of ocular injection. Furthermore, intravitreal injections also do not place the therapeutic at the desired choroid retina location. The therapeutic has to migrate through the vitreous to reach the posterior segment tissues (choroid-retina), which can potentially lead to loss of the therapeutic, either due to clearance or degradation. Besides the above mentioned topical and intravitreal routes, periocular routes including subtenon and subconjunctival routes have also been tried to deliver drugs to the back of the eye.

The periocular routes place the therapeutic agent close to the sclera and the therapeutic migrates through the sclera, reaching the choroid retina region. The risks associated with the intravitreal route of administration are overcome by the periocular routes (Raghava,

Hammond et al. 2004). Nevertheless, periocular routes are less efficient than intravitreal route in delivering the drug to intraocular tissues such as choroid(Robinson, Lee et al.

2006). Thus the foremost challenge in ocular drug delivery remains to develop a more effective route of delivery for the treatment of posterior segment disorders that can deliver therapeutics at the site of action and have least side effects.

Recently, delivery to the suprachoroidal space (SCS) has been explored as a viable option for delivery of drugs to the back of the eye. SCS is a virtual space between the sclera and choroid and suprachoroidal injection confines the therapeutic agents adjacent to the choroid-retina region, the target tissue affected in CNV. Safety of injections into the SCS was shown by Einmahl et al. (Einmahl, Savoldelli et al. 2002) in rabbits and by Poole et al. (Poole and Sudarsky 1986) in humans. Suprachoroidal injections were found to be safe with some slight choroidal pigmentation observed by

Einmahl et al., and slight bleeding and inflammation at the injection site observed by

Poole et al. More recently, Patel et al. (Patel, Lin et al. 2011) developed a hollow microneedle system for suprachoroidal delivery and assessed the ex vivo distribution of sulforhodamine B dye and particles ranging in size from 20 to 1000 nm. Suprachoroidal delivery might allow superior drug delivery in the back of the eye tissues, without the adverse effects associated with intravitreal delivery.

Despite the extensive research in suprachoroidal delivery of therapeutics for the posterior segment disorders, there are no investigations comparing it to periocular

injections. Further, there is only one study comparing suprachoroidal and intravitreal routes of delivery (Olsen, Feng et al. 2011). With the high blood flow in choroid (Polska,

Polak et al. 2004), drug molecules are expected to clear very fast following suprachoroidal injections. Therefore, a direct comparison of different routes of ocular delivery will help establish the relative advantage of suprachoroidal delivery. The third aim of this research was to demonstrate suprachoroidal injection in a rat model for the first time and compare the pharmacokinetics of suprachoroidal injection with intravitreal and posterior subconjunctival injections using noninvasive ocular fluorophotometry.

In our study we observed that suprachoroidal administration enhanced targeting by providing higher drug levels at the target site, the chorio-retinal tissues, compared with IVT injection. Also targeting the suprachoroidal space is also expected to decrease exposure of non-target tissues. This would be advantageous when delivering highly potent drugs, such as steroids, that can cause side effects. These potential clinical benefits make suprachoroidal delivery particularly attractive. However, drugs including small molecules and macromolecules have been shown to exhibit very short half-lives following suprachoroidal injection (Patel, Lin et al. 2011). In contrast, polymeric nanoparticles and microparticles remained in the suprachoroidal space for months (Patel,

Berezovsky et al. 2012). Patel et al suggested that a sieving effect is associated with clearance from the suprachoroidal space, wherein choroidal capillary walls have been shown to exclude molecules larger than 3 to 6 nm and sclera excluding particles larger than 500 nm. The sieving effect allows molecules as large as approximately 10 nm to pass but blocks particles with a diameter as small as 20 nm.

Clearance from the suprachoroidal space can be primarily attributed to capillary drainage by uptake into choroidal capillaries. The sieving effect suggested that biodegradable delivery systems encapsulating therapeutics could be used for sustained delivery to the suprachoroidal space. Thus, there is a need to develop a slow release system for therapeutic proteins such as bevacizumab and transferrin-tumstatin, in order to maintain therapeutically relevant concentrations over extended periods. Numerous sustained delivery approaches including liposomes (Kim, Choi et al. 1998) and polymeric micro- and nano- particulate systems have been assessed to sustain protein drug delivery

(Genta, Perugini et al. 2001; Sinha and Trehan 2003). However, most of these techniques fail to demonstrate release of stable proteins over long durations. It is because most techniques for preparing polymeric delivery systems adversely affect protein stability.

The formulation of sustained delivery systems such as microparticles and nanoparticles introduce organic solvent–water and air–water interfaces, which are major sources of protein denaturation and aggregation (Cleland and Jones 1996; Kim and Park

1999). Furthermore, sonication used during particle preparation can reduce protein stability(Suslick 1986). Liposomes, especially small vesicles employ sonication, which can be detrimental for protein stability (Lentz, Madden et al. 1982). In comparison to particulate systems and liposomes, gel systems are more attractive and offer a promising delivery platform due to their simplicity in formulation and solvent-free condition (Lu and Anseth 1999). In situ forming gels based on a stimuli-response are particularly attractive for ophthalmic application (Christie and Kompella 2008). To form such gels in situ, a polymeric mixture can potentially be injected through a small gauge needle in a confined space within the eye, followed by activation using a stimulus such as light. This

is beneficial since viscous gels might require large bore needles and relatively prolonged duration of injection. Injectability is of importance for ocular delivery systems, as large bore needles such as 20 or 25 gauge have higher risks of endophthalmitis and hypotony when used for ocular delivery by intravitreal injections (Kunimoto and Kaiser 2007).

Furthermore, suprachoroidal injection using a large bore needle might cause hemorrhage

(Low, Ng et al. 2009). If a drug formulation can be delivered through a 30 gauge or smaller gauge needle, the system is expected to be safer than the larger gauge needle.

Thus, the fourth aim of this research was to develop an in situ forming gel to sustain the release of stable, active bevacizumab and assess the sustained in vivo delivery in rat eyes following in situ gel formation in the suprachoroidal space.

Thus, the overall goal of this study was to test the hypotheses that 1) transferrin- tumstatin is superior to bevacizumab in treating CNV following intravitreal injection, 2) pharmacokinetics of transferrin-tumstatin can be assessed by non-invasive fluorophotometry and is safe, 3) suprachoroidal delivery is feasible in rats and the route has advantage over other routes of ocular delivery, and 4) in vivo sustained delivery of stable and active protein therapeutics is possible using an in situ forming gel system.

Specific aims for the thesis

Specific aim 1: To design, express, purify, characterize, and evaluate the efficacy of transferrin-tumstatin in vitro and in vivo in a disease model

• Task 1: To create a plasmid with transferrin-tumstatin gene and express

transferrin-tumstatin in human retinal pigment epithelial cells (ARPE-19) (a

mammalian expression system).

• Task 2: To evaluate the efficacy of transferrin-tumstatin to inhibit proliferation,

tube formation and invasion, and induce apoptosis in vitro in RF/6A (choroid

endothelial) cells.

• Task 3: To evaluate the in vivo dose dependent efficacy of transferrin-tumstatin to

inhibit CNV in a laser induced rat model and compare efficacy at an optimized

dose with bevacizumab and tumstatin.

Specific aim 2: To determine the pharmacokinetics and safety of transferrin- tumstatin in rabbits following a single intravitreal injection

• Task 1: To inject Alexa 488-conjugated transferrin-tumstatin in rabbits by

intravitreal injection and assess ocular drug levels using non-invasive

fluorophotometry.

• Task 2: To assess safety of intravitreal injection by slit lamp imaging and IOP

measurement.

• Task 3: To perform serum biochemical analysis and determine the antibody titer

produced following single intravitreal injection of transferrin-tumstatin.

Specific aim 3: To demonstrate suprachoroidal injection in a rat model and compare the pharmacokinetics of suprachoroidal injection with intravitreal and posterior subconjunctival injections using noninvasive ocular fluorophotometry

• Task 1: To perform suprachoroidal injection in Sprague Dawley (SD) rats using

India ink and confirm the success of the procedure using histology.

• Task 2: To determine the disposition of sodium fluorescein (NaF) using Fluorotron

Master™, an ocular fluorophotometer following injection in suprachoroidal space,

posterior subconjunctival region, or vitreous humor of the right eye of the SD rats. 9

• Task 3: To perform non-compartmental pharmacokinetic analysis for

suprachoroidal, posterior subconjunctival, and intravitreal routes of injection of NaF

using WinNonlin software.

Specific aim 4: To develop an in situ forming gel to sustain the release of stable, active bevacizumab (a model protein) and assess the sustained in vivo delivery in rat eyes following in situ gel formation in the suprachoroidal space

• Task 1: To develop a light activated gel using a polycaprolactone-derivatized

dimethacrylate (PCM) and hydroxyethyl methacrylate (HEMA).

• Task 2: To assess in vitro sustained release, stability, and safety of bevacizumab

from gel.

• Task 3: To assess in vivo sustained delivery and safety of bevacizumab from gel

formulations in SD rats.

CHAPTER II

INTRODUCTION

Age related macular degeneration

Age related macular degeneration (AMD) is a major cause of blindness in the

United States and other industrialized nations. AMD affects the central region of retina, the macula, and manifests mostly in elderly patients over 40 years of age (Friedman

2008). An estimated 7.2 million Americans are suffering from AMD and age acts as a major catalyst , affecting 3% of the population at 40-60 and 13% individuals > 60 years of age (Klein, Chou et al. 2011). By 2050, the number of people in the United States aged

60 years and older is expected to be 82.7 million (US Census Bureau Population

Division). With the increase in number of people > 60 years of age, cases of AMD are expected to escalate. Clinically, AMD can be divided into the dry or atrophic form and wet exudative, or neovascular form. The dry form is characterized by geographic atrophy of retinal pigment epithelium (RPE) in the macula. The wet form is characterized by neovascularization of choroidal blood vessels, known as choroidal neovascularization

(CNV). Dry AMD is a chronic disease resulting in visual impairment to some extent. In comparison, the wet form emerges rapidly progresses to severe blindness (Wong,

Chakravarthy et al. 2008). Since AMD patients initially develop dry AMD form and then wet AMD, dry AMD can be considered an antecedent for wet AMD (Ambati and Fowler

2012). Treatment of AMD is an unmet need, with Visudyne®, Lucentis®, and Eylea® as the only FDA approved therapies to treat the end-stage wet form of the disease.

Furthermore, these therapies are not very efficient in improving vision. Visudyne, a photodynamic therapy, causes visual disturbances in 22% patients and injection site adverse events such as hemorrhage and extravasation in 16% patients (Bressler 2001).

Lucentis, an anti-VEGF Fab fragment, only improves vision in 40% of the patients

(Rosenfeld, Brown et al. 2006). A phase II study with Eylea reported hemorrhage in 35% of patients (Brown, Heier et al. 2011)

Basic definition of AMD

Clinically, AMD (Figure 2.1) can vary from a few soft drusen and normal vision to CNV with legal blindness (Abdelsalam, Del Priore et al. 1999). Drusen are deposits of extracellular debris located between the the retinal pigment epithelial (RPE) and the

Bruch’s membrane (BrM) (Sarks 1980; Green and Enger 1993). Drusens are regarded as hallmarks of AMD and primarily contain apolipoproteins (Anderson, Ozaki et al. 2001) and extracellular matrix (Mullins, Russell et al. 2000). The RPE cells are believed to secrete the apolipoproteins and result in accumulation above the BrM. Histochemical analysis and proteomics of drusen have revealed many components including proteins involved in inflammation and innate immunity (Anderson, Mullins et al. 2002; Edwards,

Ritter et al. 2005). Other than protein components, carbohydrates released following the shedding of outer segments of photoreceptors are also a part of drusen (Mullins and

Hageman 1999).

AMD often progresses with a disappearance of drusen, wherein there is a loss of

RPE and thinning of the photoreceptors followed by tissue death (Klein, Ferris et al.

2008). Disappearance or fading of drusen generally follows detachment of RPE and retina (Gass 1973) in AMD patients. At the advanced stages of AMD, the onset of

choroidal neovascularization can also occur. CNV is characterized by the growth of uncontrolled blood vessels from the choroid below the macula. These new blood vessels leak blood and other components into the retina, causing visual distortion and loss of central vision (Figure 2.1).

Figure 2.1: Different stages of AMD. Early stage AMD is characterized by the presence of drusen in between the RPE and BrM. Disruption of tight junction RPE barrier and proliferation of choroidal blood vessels occurs in CNV.

Choroidal neovascularization

Choroidal neovascularization (CNV) is the uncontrolled growth of new choroidal blood vessels into the subretinal space through breaks in BrM (Pauleikhoff 2005). The disruption or break in BrM, caused by conditions such as myopic degeneration, ocular histoplasmosis, angioid streaks, punctate inner choroidopathy, or choroidal rupture caused by trauma, predisposes the eye to CNV (Norton, Gass et al. 1965; Gass 1977).

Other than alterations in BrM, CNV can also be initiated by reduction in blood flow, accumulation of metabolic by-products, and oxidative stress (Green 1999; Ambati,

Ambati et al. 2003). In response to breakage in BrM or other mentioned stresses, the RPE and retina produce factors that induce activation of choroid endothelial cells, resulting in their proliferation and migration and subsequent formation of new blood vessels. The final pathological stage following CNV formation and leakage of blood and other proteins is formation of disciform scar, which leads to permanent vision loss (Gass 1971).

The precise mechanism for the stimulation of CNV is unknown, though it is understood that the balance between circulating endogenous pro-angiogenic factors that stimulate (pro-angiogenic) and that inhibit vessel growth (anti-angiogenic) is lost

(Folkman 1995) (Folkman 1971). In normal eyes, inhibitory factors such as thrombospondin, tumstatin, angiostatin and canastatin, endostatin, and pigment epithelium-derived factor prevail and vessels remain quiescent. In contrast, in CNV, neovascularization occurs because of decrease in the production of anti-angiogenic factors and/or increase in the production of pro-angiogenic stimulus. Stimulation of CNV occurs in the presence of pro-angiogenic factors like vascular endothelial growth factor

(VEGF), ﬁbroblast growth factors, interleukins, insulin-like growth factors and

angiopoietins playing a role in angiogenesis (Bhutto and Lutty 2012). Among the mentioned angiogenic factors, currently, VEGF is currently portrayed to be the most important factor leading to the angiogenesis in CNV.

Role of pro-angiogenic factors in CNV

Among the pro-angiogenic factors known so far, VEGF plays a key role in the pathogenesis of CNV. VEGF has been demonstrated to be upregulated in animal models of CNV (Shen, Yu et al. 1998). The VEGF family includes placenta growth factor,

VEGF-A,B,C,D, and E (Eriksson and Alitalo 1999) (Ferrara 1999; Persico, Vincenti et al.

1999). VEGF-A, which has been most comprehensively studied, is a 36–46 kDa protein with an N-terminal signal sequence and a heparin-binding domain. In humans, four different VEGF-A isoforms have been identified and labeled according to the numbers of amino acids present in them: VEGF 121 , VEGF 165 , VEGF 189 , and VEGF 206 . They arise from alternative splicing of mRNA (Plate and Warnke 1997). Normal retina expresses both VEGF mRNA and protein. The VEGF 121 and VEGF 165 isoforms are abundantly expressed in the choroid, RPE, retina, and iris tissues of adult monkeys and mouse eyes

(Kim, Ryan et al. 1999). VEGF receptors have been found in the neural retina of Sprague dawley rats (Gilbert, Vranes et al. 1998). In humans, VEGF mRNA and protein expression has also been confirmed in retinal ganglion, muller cells, and the choroid

(Stitt, Simpson et al. 1998).

Other than VEGF, other pro-angiogenic factors also play a major role in CNV.

During neovascularization (Figure 2.2), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMPs) promote the activity of endothelial cells to invade and migrate (Mignatti and Rifkin 1996). Expression of protease genes is induced by cytokines

and the angiogenic factors basic fibroblast growth factor (bFGF) and VEGF-A, whereas proteolysis is upregulated by activation of pro-proteinases on the one hand and by down regulation of levels of endogenous inhibitors. Activated endothelial cells express integrins such as αvβ3 and αvβ5 allowing migration through the degraded matrix, followed by proliferation of endothelial cells (Eliceiri and Cheresh 1999). Subsequently, endothelial cells of the newly formed blood capillaries create a new basement membrane.

Further stabilization of the new blood capillaries is considered to be accompanied by employment of pericytes and smooth muscle cells (Crocker, Murad et al. 1970).

Figure 2.2 : Schematic representation of angiogenesis in CNV. Abbreviations are as follows; BrM –basement membrane, VEGF- vascular endothelial growth factor, bFGF- basic fibroblast growth factor, MMPs-matrix metalloproteinases, uPA- urokinase-type plasminogen activator. Figure is drawn based on Figure 3 from Basement membranes: structure, assembly and role in tumour angiogenesis by Raghu Kalluri (Kalluri 2003)

Therapeutic strategies for CNV

Although the symptoms of choroidal neovascularization are comparatively straightforward, the pathogenic causes underlying the disease are various. Therapeutic strategies for the management of CNV are therefore not causal treatments, but generally they help to avoid further loss of vision rather than improve vision. None of the treatments employed up-to-date can cure the disease. The current treatments merely halt the progress of the disease, and that too also not to a great extent. There are the following established medical treatments (in chronological order), including anti-angiogenesis approaches that have been used to date for the treatment of CNV.

Laser photocoagulation

Photocoagulation of well defined CNV using argon laser (wavelength 488 and

514 nm) was introduced in the earlier 1970s. Further development led to the introduction of krypton lasers which emit at 647 nm. Multiple studies demonstrated the effectiveness of both argon as well as krypton lasers (Group 1994; Willan, Cruess et al. 1996; Ruiz-

Moreno and Montero 2002) in reducing the risk of visual loss in patients with CNV. One disadvantage is that it causes a dark spot in the visual field. Also, the recurrence rate is too high following laser photocoagulation.

Photodynamic therapy

As laser photocoagulation for CNV is ridden with limitations, other less destructive treatments are needed to improve the visual outcome in CNV. Verteporfin

(Visudyne®; QLT Ophthalmics, Inc) photodynamic therapy (vPDT) was introduced in the late 1990s as a treatment for CNV. Photodynamic therapy involves two steps. A photosensitive drug verteporfin , is first injected intravenously (Aveline, Hasan et al.

1994). Following injection, verteporfin is activated by nonthermal light (659 nm)

(Manyak, Russo et al. 1988). Investigators (Schmidt-Erfurth, Hasan et al. 1994) (Miller,

Walsh et al. 1995) hypothesized that photodynamic therapy may be very useful in the selective destruction of CNV without causing significant destruction to viable neurosensory retina overlying the CNV, thereby confining the lesion from growing and reducing the risk of progressive visual damage.

Macugen

Pegaptanib sodium (Macugen®), an anti-VEGF aptamer, was approved in 2004 as a treatment of all subtypes of wet AMD. Macugen is an intravitreally administered aptamer (single stranded nucleic acids) which selectively binds with VEGF 165 isoform of

VEGF-A, and prevents VEGF from stimulating the endothelial cell surface VEGF receptor (Waheed and Miller 2004).

Bevacizumab

Bevacizumab (Avastin®) is a humanized, VEGF neutralizing monoclonal antibody (Rosenfeld 2007). Bevacizumab is the first anti-angiogenic agent to be approved by the FDA for the treatment of a cancer (Ferrara, Hillan et al. 2004). Bevacizumab binds to and neutralizes all human VEGF-A isoforms. Bevacizumab is not effective in inhibiting other members of the VEGF gene family, such as VEGF-B or VEGF-C

(Ferrara, Hillan et al. 2004). In recent years, ophthalmologists have increasingly begun prescribing bevacizumab as an off-label treatment for CNV by intravitreal route of injection. The most apparent reasons for the acceptance of intravitreal bevacizumab include the affordable, low cost of intravitreal bevacizumab (Rosenfeld, Fung et al. 2005;

Rosenfeld, Moshfeghi et al. 2005), and the similarity in efficacy to Lucentis

(ranibizumab, Genentech, Inc).

Ranibizumab

In 2006, ranibizumab (Lucentis®), a recombinant Fab fragment of VEGF-A, neutralizes all isoforms of VEGF-A (Rosenfeld, Brown et al. 2006) was approved by the

United States Food and Drug Administration (US FDA) for the treatment of subtypes of neovascular AMD (Rosenfeld, Brown et al. 2006). Ranibizumab is an antibody fragment developed from the same antibody as bevacizumab and by the same company,

Genentech. Lucentis is currently regarded as treatment gold standard for the therapy of

AMD. Two phase III clinical trials have demonstrated that severity of vision loss can be reduced by monthly intravitreal injection of ranibizumab (Rofagha, Bhisitkul et al. 2013).

Aflibercept

In 2011, US FDA approved Eylea® ( Regeneron,Inc) is a fusion protein created from the immunoglobulin (Ig) binding domain of VEGF receptor 1 and Ig binding domain of VEGF receptor 2, fused to the Fc region of human IgG1. It binds to all

VEGF-A isoforms, VEGF-B, and placental growth factor (PlGF) (Papadopoulos, Martin et al. 2012). Unlike bevacizumab and ranibizumab, aflibercept binds to PlGF and VEGF-

B, in addition to all isoforms of VEGF-A. Similar to VEGF, PlGF has been shown to contribute to CNV (Rakic, Lambert et al. 2003).

Drawbacks of current therapeutic strategies of CNV

Pro-angiogenesis inhibitors such as bevacizumab that target the VEGF signaling pathways have demonstrated noteworthy therapeutic efficacy in many human cancers.

However, the benefits are at best temporary and are followed by a return of tumor growth

and progression. Data support a hypothesis that two modes of unconventional resistance can be responsible for this outcome: evasive resistance, wherein angiogenic tissues adapt to evade the specific pro-angiogenic blockade; and inherent or pre-existing resistance to anti-VEGF therapy (Bergers and Hanahan 2008).

In an attempt to understand how neovascularization is maintained and tumors are able to circumvent the anti-VEGF treatment, Chung et al. (Chung, Wu et al. 2013) showed that pro-inflammatory cytokine, interleukin-17 (IL-17), dependent network is initiated in cells that are treated with anti-VEGF therapies. Thus, a VEGF-independent angiogenesis occurs in cells. Evasion of anti-VEGF therapy has also been associated with recruitment of bone marrow derived cells. Infiltration of CD11b+Gr1+ pro-angiogenic immature myeloid cells has been noted in refractory tumors compared to those sensitive to anti-angiogenic therapies (Shojaei, Wu et al. 2007). These bone marrow derived cells can then differentiate into endothelial cells and pericytes, and also release growth factors, cytokines and matrix metalloproteinases (Du, Lu et al. 2008). The accumulation of bone marrow derived cells is commonly seen in most cancer types and is indicative of tumor progression. Bergers at al. (Bergers and Song 2005) proposed a resistance mechanism in this scenario, wherein pericytes might be having a protective effect on angiogenic blood vessels. Neovascularization is also promoted by components of angiogenic microenvironment. Tumor-associated fibroblasts, a major component of the angiogenic stroma, from tumors resistant to anti-VEGF therapy promote tumor growth by up regulating the expression of pro- angiogenic genes, including platelet-derived growth factor (PDGF), angiopoietin-related protein 2 (Angptl2), and cyclooxygenase-2 (COX2)

(Crawford, Kasman et al. 2009).

Besides the upregulation of other pro-angiogenic pathways following anti-VEGF treatment, some inherent, pre-existing resistance can also be responsible for the lack of anti-VEGF therapy efficacy (Bergers and Hanahan 2008). In a clinical trial with 716 patients, VEGF inhibition by ranibizumab did not improve vision in 60% of wet AMD patients (Rosenfeld, Brown et al. 2006). More recently, it was determined that ~20% of the patients treated with anti-VEGF therapy lose vision over time (Brown, Heier et al.

2011). A further ~ 20% may lose the initial visual improvements achieved with anti-

VEGF treatments (Brown, Heier et al. 2011). Similarly an analysis of human breast cancer biopsies exposed that late-stage breast cancers expressed a lot of non-VEGF, pro- angiogenic factors such as FGF2 (Relf, LeJeune et al. 1997). Thus, it can be hypothesized that the pre-existence of FGF2 and the other pro-angiogenic factors in late-stage cancers could enable the progression of angiogenesis despite being treated with bevcizumab, such that inhibition of VEGF signaling does not affect angiogenesis.

All the above studies strongly indicate that other pro-angiogenic pathways may replace VEGF as the disease progresses and eventually, anti-VEGF therapies are not of any use. Therefore, further investigations are needed to develop more potent and biological relevant therapeutics that can be affect diverse pathways.

Endogenous inhibitors of CNV

In addition to the pro-angiogenic factors such as VEGF, which promote angiogenesis, tissues and extracellular matrix also have anti-angiogenic proteins, which have the capability to prevent angiogenesis by inhibiting the signaling mechanisms activated by angiogenic factors (Zhang and Ma 2007; Boosani, Nalabothula et al. 2010).

Many endogenous anti-angiogenic proteins have been studied and some of them are

found to be present in the ocular tissues or reach the ocular tissues via blood circulation, where they exhibit anti-angiogenic properties and eventually regulate CNV. As previously described, angiogenesis is tightly controlled by a strict balance between pro- angiogenic and anti-angiogenic factors. In quiescent healthy tissues, anti-angiogenic factors predominate and prevent angiogenesis (Bouck 2002). In contrast, in a variety of pathological states such as CNV, neovascularization occurs because of decreased levels of inhibitors (Wang, Gottlieb et al. 2009) and/or increased production of pro-angiogenic factors such as VEGF (Hoeben, Landuyt et al. 2004). The significance of the imbalance has been ascertained by Bhutto et al, wherein it was confirmed by immunostaining that the levels of 3 potent anti-angiogenic endogenous inhibitors, namely endostatin, PEDF, and thrombospondin 1, were significantly reduced in Bruch's membrane in eyes with

CNV (Bhutto, Uno et al. 2008). Furthermore, it has been shown that by administration of recombinant endogenous anti-angiogenic protein such as endostatin (Marneros, She et al.

2007) and vasostatin (Sheu, Bee et al. 2009), CNV can be reduced.

In our study, we used tumstatin, an endogenous inhibitor generated from the NC1 domain of type IV collagen α3 chain (Hamano, Zeisberg et al. 2003). Tumstatin plays a pivotal role in inhibiting blood vessel formation by binding to αVβ3 integrins, hindering the proliferation of endothelial cells and also by inducing endothelial cell apoptosis

(Maeshima, Colorado et al. 2000).

Inhibition of angiogenesis by tumstatin

Tumstatin is a fragment derived from the NC1 domain of α3 collagen IV following proteolysis by MMP-9 (Hamano, Zeisberg et al. 2003). Tumstatin has been shown to inhibit proliferation in endothelial and cancer cells (Han, Ohno et al. 1997;

Maeshima, Colorado et al. 2000) in vitro. Tumstatin also inhibits angiogenesis in xenograft models of prostate cancer (Maeshima, Colorado et al. 2000; Maeshima,

Manfredi et al. 2001), renal cell carcinoma (Eikesdal, Sugimoto et al. 2008), oral squamous cell carcinoma (Chung, Son et al. 2008). Further, Maeshima et al (Maeshima,

Manfredi et al. 2001) and Eikesdal et al. (Eikesdal, Sugimoto et al. 2008) identified the fragments that are responsible for the efficacy of tumstatin. Fragments of tumstatin has shown efficacy in gastric carcinoma (He, Jiang et al. 2010), ovarian cancer (Zhang, Sui et al. 2008), hepatocellular carcinoma (Goto, Ishikawa et al. 2008), glioma (Kawaguchi,

Yamashita et al. 2006), and colon cancer (Cao, Peng et al. 2006). Apart from the mentioned cancers, tumstatin also plays a role in the progression of bronchopulmonary carcinomas (Caudroy, Cucherousset et al. 2004). Recently, reduced tumstatin mRNA in tumor tissues was correlated with poor patient outcome in non-small cell lung carcinoma

(Luo, Ming et al. 2012).

Molecular mechanism of tumstatin has shown to encompass binding to integrin

αvβ3 and subsequent inhibition of protein synthesis (Cooke and Kalluri 2008). Binding to integrin αvβ3 prevents the activation of down-stream pathways of protein translation and angiogenesis effect (Sudhakar, Sugimoto et al. 2003). In a secondary mechanism, tumstatin is also reported to bind to integrin α3β1 inhibits hypoxia induced COX-2 expression in endothelial cells (Boosani, Mannam et al. 2007). 23

However, tumstatin has shown a very high dose requirement of 4.4-8.8 mg/kg every 2 days for treatment of cancer (He, Jiang et al. 2010), which indicates that translation of the therapeutic efficacy in humans will be very difficult due to the high dose. Therefore, more efficacious derivatives of tumstatin are required to improve its chances of reaching the clinic as a therapeutic.

Transferrin-tumstatin, a novel fusion protein for CNV

Transferrin-tumstatin (Tf-T) has been created in this study to improve upon the efficacy of tumstatin. Tf-T is expected to have many advantages over the current standard of care for CNV (ranibizumab and bevacizumab) and is also anticipated to perform better than the parent molecule tumstatin. The fusion protein takes advantage of the transferrin receptor-mediated uptake to enhance delivery of proteins across cellular barriers. Further, transferrin can be utilized to target the protein to proliferating endothelial cells, wherein the transferrin receptor is overexpressed (Sutherland, Delia et al. 1981). Tumstatin also has capability for cell specific delivery, with the tumstatin receptor, αVβ3 integrin only being found on activated endothelial cells and not on normal blood vessels(Friedlander, Theesfeld et al. 1996). Thus, tumstatin acts as a selective target towards activated endothelial cells, without affecting physiological processes such as wound healing (Hamano, Zeisberg et al. 2003). Thus, transferrin- tumstatin, as a therapeutic agent, can potentially treat neovascularization and restore vision loss.

Current techniques for drug delivery to the posterior region of the eye

Drug delivery to the posterior segment of the eye is useful in treating various disorders including CNV, as choroid retina region is the primary target for most of the vision threatening disorders. Delivery of drugs in therapeutically relevant concentrations to the retina continues to remain a challenge for pharmaceutical scientists because of the ocular anatomical and physiological barriers. The challenges further aggravate if frequent long term therapy is required as is the case with diseases like diabetic retinopathy and

CNV. Topical instillation of the therapeutic is the most commonly used route for ocular administration and this route has been successful in treating diseases afflicting the anterior segment of the eye. However, topical route of delivery is inundated by limitations. One of the major problems encountered with the topical administration of therapeutics is the poor bioavailability of the drug, with only ~ 5% (Urtti, Pipkin et al.

1990) of the drug being absorbed into the aqueous humor. Multiple obstacles, such as short precorneal residence time and the presence of epithelial barriers, are primarily responsible for the poor bioavailability. Topically applied drug formulations were found to have a half-life of ~ 1.3 minutes (Meadows, Paugh et al. 2002). Cornea (Klyce and

Crosson 1985) and conjunctiva (Kompella, Kim et al. 1993) are tight epithelial barriers that limit drug absorption due to the presence of tight junctions. Also, the drug that crosses conjunctiva can be cleared by episcleral blood supply. Due to the presence of such challenging barriers, eye drops fail to efficiently deliver a drug to the ocular tissues.

The extent of delivery is expected to be much lower in the posterior tissues such as retina and choroid when compared to the anterior segment. Systemically administered therapeutic agents reach the retina to a limited extent due to the presence of blood ocular

barriers, which include the blood-aqueous barrier and the blood-retinal barriers

(Kompella, Kadam et al. 2010). The blood aqueous barrier is formed by the ciliary bodies and iris vessels. The blood-retinal-barriers include the RPE and the endothelium of the retinal blood vessels. Due to these barriers, attainment of therapeutic effect requires high systemic doses, which can produce harmful side effects since majority of the drugs intended for the eye were originally developed for systemic use (Davidson and Rennie

1986).

Despite the significant prevalence of posterior segment disorders such as CNV, delivery to the diseased tissues is limited primarily to invasive methods such as repeated intravitreal injections. Since the first report on intravitreal injections in 1944, this mode of administration found wide clinical application for treating choroid retinal disorders.

Intravitreal injections can provide high drug concentrations to the back of the eye. While this route of delivery provides sufficient drug levels for efficacy, repeated intravitreal injections have been associated with complications like retinal detachment and endophthalmitis and thus have poor patient compliance (Arevalo, Maia et al. 2008;

Mezad-Koursh, Goldstein et al. 2010). Furthermore, intravitreal injections can result in high drug levels, potentially causing retinal toxicity, as observed with gentamicin

(Campochiaro 2000). However, decreased elimination half-life of high molecular weight compounds such as bevacizumab and ranibizumab for the vitreous makes intravitreal route ideal for these therapeutics (Bakri, Snyder et al. 2007; Bakri, Snyder et al. 2007).

However, such drugs still require repeated intravitreal administrations in treating chronic disorders, which might exacerbate the risks.

Suprachoroidal delivery for posterior region of the eye

Suprachoroidal space is a virtual space located between the sclera and choroid. By injecting in the suprachoroidal space, targeting to the choroid retina region can be achieved. Injection of therapeutics in the suprachoroidal space can place the therapeutic in close vicinity to the target tissues for disorders such as CNV. For this reason, the suprachoroidal space signifies a likely new site of administration for treatment of posterior segment diseases. Evidence of injections to the suprachoroidal space was first described by Einmahl et al. (Einmahl, Savoldelli et al. 2002) in 2002 using a rabbit model. Poly-ortho ester was injected into the suprachoroidal space and it was demonstrated that the polymer was localized in the space for 3 weeks. In 2006, Olsen et al. reported the use of a cannula in pigs to deliver into the suprachoroidal space (Olsen,

Feng et al. 2006). Olsen et al. demonstrated safety of the delivery route and also established sustained delivery. More recent studies (Olsen, Feng et al. 2011) have determined the kinetics of macromolecules such as bevacizumab injections in the suprachoroidal space. Further, Patel et al. (Patel, Berezovsky et al. 2012) demonstrated that though suprachoroidal route is advantageous in delivering at the target tissues

(choroid and retina) even macromolecules such as bevacizumab are cleared very rapidly.

The cumulative results of the discussed studies indicate that even though suprachoroidal route is very effective in delivering high concentrations of drug at choroid, sustained delivery systems are of utmost need to sustain the delivery even for macromolecules such as bevacizumab.

Figure 2.3 Schematic representation of intravitreal and suprachoroidal route of injection in the eye.

Sustained delivery systems for posterior region of the eye

Various sustained delivery systems have developed and approved for use in diseases such as CNV. These polymeric delivery systems are fabricated to attain prolonged therapeutic drug levels in the target choroid retina tissues that are not readily accessible by conventional delivery techniques. Sustained delivery systems also limit the side effects from systemic drug exposure and repeated injections as well as improve patient compliance (Yasukawa, Ogura et al. 2006; Wadhwa, Paliwal et al. 2009). The sustained release systems can be classified as biodegradable and non-biodegradable, depending upon the nature of the polymer used (Table 2.1).

Table 2.1 FDA approved sustained delivery systems for delivery to the posterior region of the eye.

Delivery Drug (Trade Disease Company Route Reference approach name) target Diabetic macular edema, (Haller, Implant Dexamethasone ® Allergan Intravitreal uveitis, Dugel et (biodegradable) (Ozurdex ) post- al. 2009) cataract surgery (Kedhar Ganciclovir Bausch & CMV Intravitreal and Jabs (Vitrasert ®) Lomb retinitis 2007) Implant (non- Chronic Fluocinolone Bausch & biodegradable) uveitis, (Mohamm acetonide Lomb/ Intravitreal diabetic ad, Sweet (Retisert ®, ) pSivida macular et al. 2007) Ltd. edema

Ozurdex ®, a sustained release implant of dexamethasone for delivery to the posterior segment of the eye has been developed by Allergan Inc,CA. Ozurdex® provides sustained release for up to six months. It is advantageous to have a sustained delivery system for dexamethasone because the drug, though very potent, has a very short intravitreal half-life (Kwak and D'Amico 1992). Ozurdex ® is loaded with 0.7 mg of dexamethasone in a poly (lactic-co-glycolic) acid copolymer. The implant is designed to release dexamethasone in two phases, wherein the first two months have higher doses, and the remaining months have low maintenance dose. Vitrasert® (Bausch and Lomb,

Inc.) is a non-biodegradable ganciclovir implant developed for the treatment of cytomegalovirus retinitis. Ganciclovir is loaded within a reservoir, encased by poly vinyl alcohol/ ethyl vinyl alcohol membrane. Drug diffuses out according to zero-order release kinetics following the entry of water in the delivery system (Yasukawa, Ogura et al.

2006). The device provides sustained release of ganciclovir for 5–8 months. Vitrasert® is ideal for treatment of cytomegalovirus retinitis that pose an immediate risk to vision, and combination treatment with oral valganciclovir can be used to prevent the spread of the infection to the other eye (Kedhar and Jabs 2007). Retisert® is a non-biodegradable implant loaded with fluocinolone and coated with silicone and PVA (Conway 2008;

Kiernan and Mieler 2009). The device has an initial drug delivery rate of 0.6 mg/day and reaches a steady-state delivery rate of 0.3–0.4 mg/day over roughly 30 months.

Despite the widespread use of the above mentioned sustained delivery systems for small molecules, sustaining the release of protein therapeutics is significantly more difficult. The molecular size of proteins is far higher than that of small molecules.

Furthermore, proteins have secondary and tertiary structures, which make them very susceptible to degradation.

Sustained release systems for proteins

Frequent injections, primarily due to their tissue impermeability and short in vivo life of macromolecules, are required for macromolecule therapy. For example, ranibizumab requires intravitreal injection every month for treatment of CNV. Retinal detachment, hemorrhage at the site of injection, and risk of infection are complications of repeated injection of ranibizumab (Regillo, Brown et al. 2008). Furthermore, repeated injections result in poor patient compliance. Sustained release delivery systems can potentially reduce dosing frequency and decreasing adverse side effects.

To achieve the daunting task of sustaining the release of protein therapeutics, various formulation strategies have been examined. Delivery of proteins by biodegradable microspheres is the most preferred one till today because of the biocompatibility and biodegradability of polymers used for their preparation. The aliphatic polyesters are the most widely investigated biodegradable polymers for protein delivery and are based on poly (lactic acid) (PLA), poly- (glycolic acid) (PGA), and their copolymers, poly(lactic acid-co-glycolic acid) (PLGA). Biodegradable polymers that degrade in vivo to generate non-toxic byproducts are ideal for clinical use. Furthermore, release rate for these delivery systems can be tailored by altering the composition of the biodegradable polymer. However, each of the delivery systems has its own drawbacks and can lead to instability of the protein therapeutic loaded within the system. For example, efforts to sustain delivery using PLGA nano/microparticles is thwarted by the instability faced by the protein at water/dichloromethane interface (Sah 1999; Sah 1999).

Proteins are easily denatured when minor changes of pH and temperature takes place.

Therefore, a current need is for development of sustained delivery systems for proteins that can sustain release of active and stable proteins over long durations.

In situ cross-linking gel based systems are attractive for formulating sustained release delivery systems for proteins. In situ forming gels can potentially be prepared by avoiding the use of organic solvent exposure to protein drugs. In situ gels have been prepared using polymers such as carbopol (polyacrylic acid), poloxamers (poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) triblock copolymers) and poly methacrylic acid. The protein can be mixed with the in situ gel forming polymers in its solution or fluid form prior to administration. Following administration, gel forming polymer fluid converts to gel immediately after injection into the body and encapsulates the proteins within it in the form of a depot. In vivo gelling can be performed by change in temperature (Cai, Liu et al. 2005), by shining UV light (Mellott, Searcy et al. 2001), and chemical cross-linking (Yamamoto, Takahashi et al. 2006).

CHAPTER III

A NOVEL TUMSTATIN FUSION PROTEIN FOR CHOROIDAL

NEOVASCULARIZATION

Abstract

Choroidal neovascularization (CNV) is the leading cause of visual impairment and vision loss in patients 60 years or older. Inhibition of VEGF has been developed as a viable therapeutic strategy but with limited success. Here we explore a second mechanism for blocking angiogenesis based on the anti-angiogenic collagen IV fragment termed tumstatin. We fused tumstatin to the C-terminus of transferrin to create a new protein; transferrin-tumstatin (Tf-T). Transferrin receptors are known to be upregulated during angiogenesis, providing an additional level of targeting. Transferrin is also rapidly and efficiently taken up by receptor mediated endocytosis and then secreted preferentially on the basolateral side of the retinal pigmented epithelium where blood vessels are preferentially found. Additionally, cytoplasmic localization of Tf-T may allow the protein to function by additional mechanisms. Here we compared purified tumstatin and Tf-T to the anti VEGF antibody bevacizumab, in their abilities to block proliferation, tube formation, and cell migration and induce apoptosis. We found that Tf-T was superior to tumstatin and bevacizumab in blocking proliferation, tube formation, and migration. At the doses examined, tumstatin and Tf-T induced apoptosis while bevacizumab did not.

Tf-T was found to be readily taken up by cells while tumstatin was not. In a rat CNV model we found that a single intravitreal injection of Tf-T was superior to both tumstatin

and bevacizumab in decreasing lesion size in the disease model. We conclude that the addition of the transferrin domain to tumstatin greatly increases its anti-angiogenic properties.

Introduction

Choroidal neovascularization (CNV) refers to the uncontrolled growth of choroidal vasculature which can lead to severe vision loss in diseases such as pseudoxanthoma elasticum (Finger, Charbel Issa et al. 2009), angioid streaks (Roth,

Estafanous et al. 2001), histoplasmosis, punctuate inner choroidopathy (Gass 1997) and, most significantly wet age related macular degeneration (AMD)(Kaufman 2002). Wet

AMD occurs when the deposition of drusen (complement components, lipids, and apolipoproteins) (Lu and Adamis 2006) causes confined ischemic regions resulting in hypoxia. Hypoxia leads to an increase in the secretion of vascular endothelial growth factor (VEGF), which activates choroidal endothelial cells to secrete matrix metalloproteinases (MMP) (Ottino, Finley et al. 2004). Metalloproteinases degrade the extracellular matrix (Stetler-Stevenson 1999), thereby allowing the proliferation of endothelial cells and their migration towards the retina. The effect of MMP eventually results in the development of new blood vessels, or CNV (Spilsbury, Garrett et al. 2000), which can cause retinal detachment and hemorrhage (Lu and Adamis 2006) and the formation of sub retinal lesions (Liutkeviciene, Lesauskaite et al.) due to blood and lipid leakage. Once manifested, wet AMD related CNV is a major cause of vision loss in the elderly population of United States (Jager, Mieler et al. 2008).

Treatment of CNV is currently limited to a fraction of the patient population and focuses on restraining the detrimental role of VEGF in vascular hyperpermeability and

new blood vessel formation. Currently, ranibizumab (Lucentis TM ) and aflibercept

(Eylea TM ) are the only two anti-VEGF agents that have been approved to treat CNV.

Other than the two mentioned biological therapeutic agents, bevacizumab (Avastin™), the parent antibody from which ranibizumab was derived, is also being explored as an off label treatment for CNV (Tatar, Yoeruek et al. 2008). However, these therapies have shown limited success (Bashshur, Bazarbachi et al. 2006; Stone 2006; Bakri, Snyder et al. 2007; Bakri, Snyder et al. 2007; Bashshur, Haddad et al. 2008) for treating vascular leakage and new blood vessel growth associated with CNV. In a clinical trial of ranibizumab with 716 patients, only 40% of the patients showed an improvement in visual acuity, with no patient being completely restored to 20/20 vision (Rosenfeld,

Brown et al. 2006). Further, ranibizumab improves vision by only 7 letters (Chappelow and Kaiser 2008) in the eye chart and it does not improve vision in 60% of wet AMD patients (Rosenfeld, Brown et al. 2006). Additionally, anti-VEGF treatments can impair normal physiologic processes such as wound healing (Verheul and Pinedo 2007), photoreceptor survival, and maintenance of choroid capillary bed (Rodrigues, Farah et al.

2009). Further, anti-VEGF therapies may result in increased rates of thromboembolic events (Liew and Mitchell 2007). Repeated injections of anti-VEGF agents may also increase intraocular pressure (IOP) in patients (Goodwin, Braden et al. 1998). Further, the current treatments of wet AMD have not been able to show efficacy in regressing angiogenesis that has already occurred due to abnormal blood vessel growth. This is due to the lack of apoptotic activity of the current anti-VEGF agents.

Tumstatin is an endogenous angiogenesis inhibitor derived from the C terminus non-collagenous domain (NC1) of collagen IV present in the basement membrane

(Hamano, Zeisberg et al. 2003). It was discovered by a team led by Dr. Raghu Kalluri at

Harvard medical school (Maeshima, Colorado et al. 2000). Tumstatin plays a pivotal role in inhibiting blood vessel formation by binding to αVβ3 integrins, hindering the proliferation of endothelial cells and also by inducing endothelial cell apoptosis

(Maeshima, Colorado et al. 2000). In the absence of any pathological condition, a strict balance is maintained between angiogenic (such as VEGF) and antiangiogenic molecules

(such as tumstatin). During hypoxia and ischemia, the balance between angiogenic and antiangiogenic molecules is perturbed, thereby causing neovascularization.

Transferrin-tumstatin is expected to have many advantages over ranibizumab and bevacizumab and is anticipated to perform better than these antibodies. Transferrin receptor-mediated uptake can be utilized for enhancing delivery of proteins across cellular barriers. Further, transferrin receptor is expressed more in proliferating cells

(such as choroid endothelial cells) than quiescent cells (Sutherland, Delia et al. 1981), thereby allowing preferential delivery to proliferating cells. The other part of our fusion protein, tumstatin also has a cell specific delivery, with the tumstatin receptor, αVβ3 integrin only being found on activated endothelial cells and not on normal blood vessels

(Friedlander, Theesfeld et al. 1996; Luna, Tobe et al. 1996; Hampton 2003). Thus, tumstatin provides a path to target activated endothelial cells, without affecting physiological processes such as wound healing (Hamano, Zeisberg et al. 2003).

Tumstatin has been reported to cause apoptosis in endothelial cells (Maeshima, Colorado et al. 2000), and apoptosis caused by tumstatin can cause regression of proliferating blood vessels (Esipov, Beyrakhova et al. 2012) and be advantageous in restoring normal

vision. Thus, transferrin- tumstatin, as a therapeutic agent, can potentially treat neovascularization and prevent vision loss.

The purpose of this study is to enhance the efficacy of tumstatin by creating a new

Tf-T fusion protein and studying its efficacy in CNV. Our approach to create transferrin- tumstatin fusion protein not only retained the activity of tumstatin but also enhanced it several fold. This article reports the preparation and characterization of transferrin- tumstatin and its in vitro efficacy in comparison to tumstatin and bevacizumab. Further, it reports the efficacy of the fusion protein in in vivo in a rat CNV model.

Materials and methods

Materials

Transwell® filters (0.4µm pore size) were purchased from Corning Inc., NY.

Bovine serum albumin and MTT reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide were purchased from Sigma Aldrich, MO. BD Matrigel Matrix

Growth Factor Reduced was purchased from BD Biosciences, CA. DNA ladder,

Lipofectamine® 2000 reagent and DAPI stain was purchased from Invitrogen

Corporation, CA. The restriction enzymes used were purchased from New England

Biolabs, MA. The QuikChange® Site-Directed Mutagenesis Kit was purchased from

Agilent Technologies, CA. Transferrin gene, choroid endothelial cells (RF/6A), and the

RF/6A cell media were purchased from American Type Culture Collection, VA.

QIAGEN® plasmid Giga kit was purchased from QIAGEN Inc., CA. TALON® metal affinity resin (Catalogue # 635502) was bought from Clonetech Laboratories, Inc., CA.

BCA® Protein Assay Kit was purchased from Pierce Biotechnology, Inc. IL (Catalogue

# 23225). Pre-cast gels, 4-20% Ready Gel Tris-HCl® were purchased from Bio-Rad

laboratories, Inc., CA and EZ Run® prestained protein ladder was obtained from Fisher

Scientific, PA.

Construction of plasmids

Four different constructs were prepared for the study, containing the following cDNAs, namely, (a) tumstatin (b) tumstatin-EGFP (c) Tf-T (d) Tf-T-EGFP. All primers were purchased from Integrated DNA Technologies Inc., CA for use in this experiment.

All plasmids were grown using the DH5 α strain of E.Coli bacteria and amplified using

QIAGEN® plasmid Giga kit. Tumstatin cDNA was a gift from Dr. Sudhakar Aku

Yakkanti. Tumstatin cDNA was PCR amplified and ligated into PSecTag2B vector using

BamH1 and Xho1 restriction sites. EGFP gene was ligated using Hind III and BamH1 restriction sites. The IgK secretory sequence in the plasmid was swapped for transferrin by first digesting the construct with Nhe1 plus Sfi1 to remove the IgK sequence.

Transferrin was PCR amplified and cloned into the linearized vector using Nhe1 and Sfi1 restriction sites. A stop codon was introduced at the end of the gene construct (C terminus of tumstatin) using site directed mutagenesis.

Production and purification of tumstatin and Tf-T protein

Human retinal pigment epithelial (ARPE) cells (passage # 24) were grown in a 12 well plate to 80 % confluency. Amplified Tf-T plasmid (cloned in PSecTag2B vector) was diluted in DMEM/F12 media and mixed with lipofectamine reagent. The plasmid solution was transfected in ARPE cells and the cells were incubated at 37°C in a CO 2 incubator for 24 hours. At the end of 24 hours, media was collected and purified using ion exchange chromatography. Ion exchange chromatography was carried out using

AKTA FPLC (GE Healthcare life sciences, PA) fitted with a cationic SP sepharose bead

filled column. Protein purification was assessed using SDS PAGE and size exclusion chromatography. For SDS PAGE, 5 µg of protein was mixed with 4x loading dye and boiled for 5 minutes. Samples were run on 4–20% gradient SDS-PAGE gel (Bio-Rad,

Hercules, CA). Size exclusion chromatography (SEC) was performed using a Bio SEC-3 column (Agilent Technologies, Inc, CA) attached to a Waters HPLC. The Waters HPLC system consisted of a Waters 600S controller, Waters TM 616 solvent delivery pump,

Waters 717 plus auto-injector and a Waters 996 PDA detector. Mobile phase used for

SEC was phosphate buffer saline (PBS) pH 7.4. The sample volume injected was 25µl.

Western blot of Tf-T protein

A 10µl solution of 4x loading dye was added to 30µl of 1mg/ml Tf-T protein solution and heated at 100°C for 5 minutes and further centrifuged for 5 minutes at

13000g. Protein was loaded onto a 4-15% Tris-HCl gel and the gel was run for 45 minutes at 30 amperes. The protein was transferred out of the gel onto a PVDF membrane for 3 hours at 100 mA. The membrane was further blocked with the blocking buffer containing non fat dry milk at room temperature for 2 hours. After 2 hours, blocking buffer was discarded and the primary antibody (anti-transferrin, AB10211,

Abcam) was diluted 1:1000 and added to transfer blot. The blot was left at 4°C overnight.

Secondary antibody (NA931V,ECL mouse IgG, GElifesciences) was added to the blot and incubated at room temperature for 1 hour the next day. ECL detection kit was used to develop film. The membrane was exposed to x-ray films for 1 minute and the film was developed to get the picture.

Cell proliferation assay

An MTT assay was used to assess the proliferation of RF/6A cells under the effect of Tf-T, tumstatin, and bevacizumab protein (concentrations ranging from 1.9 – 250nM).

Choroid endothelial (RF/6A) cells (passage# 7) were plated in a 96-well plate at a seeding density of 20,000 cells/well and allowed to adhere to the well for 24 hours in the presence of serum containing media. After 24 hours, protein solutions of Tf-T, tumstatin, and bevacizumab were prepared in serum free media containing 50 ng/ml VEGF and added to the wells. Control wells contained 50 ng/ml VEGF. The cells were kept at 37°C in a 95 % air / 5% CO 2 incubator for 24 hours. After 24 hours the media was removed from all wells and replaced with 200 µl of fresh serum free media. MTT reagent (Sigma

Aldrich, MO) (20 l of 5 mg/ml MTT dissolved in PBS pH 7.4) was added to each well and incubated at 37°C for 3 hours. The medium was aspirated out and the formazan crystals formed were dissolved in 200 l of DMSO. After adding DMSO, the crystals dissolved and the absorbance was measured at 570 nm using a microplate reader. The wells containing VEGF only were used as a control (arbitrarily given the value of 100% proliferation) to calculate the reduction in cell proliferation in other wells. Protein concentrations tested were 1.9 – 250 nM for the three proteins.

Tube formation assay

Matrigel® was thawed at 4°C overnight. A 48-well plate was prepared for the tube formation assay by spreading 75µl of the thawed matrigel on the bottom of each of the wells. The plate was kept at 37°C for 30 minutes to polymerize the matrigel. RF/6A cells (6000 cells) along with the protein solutions of Tf-T, tumstatin, and bevacizumab

(concentrations ranging from 1.9 – 250nM) were prepared in serum free media and

transferred to the 48-well plate containing the matrigel. The final volume in the well was

250 µl. The plate was kept at 37°C for 18 hours in a 95% air/5% CO 2 incubator. Tube formation was analyzed using a light microscope (Olympus BX41 laboratory microscope) fitted with a camera (Diagnostics instruments Inc.) at 10X magnification.

Tubes in each field were counted and plotted.

Cell invasion assay

Cell migration assays were performed using a Matrigel invasion chamber (8 m pore size, Becton Dickinson, MA). A suspension of 50,000 RF/6A cells and protein solutions of Tf-T, tumstatin, and bevacizumab (concentrations ranging from 1.9 –

250nM) in 0.5 ml of serum free media was added to the upper chamber. The bottom chamber was filled with 1 ml of 10 ng/ml VEGF solution in serum free media. Chambers were incubated at 37°C for 24 hours in a 95% air/5% CO 2 incubator. After the required incubation time, non-invading cells were removed from the upper surface of the membrane by scrubbing with a sterile cotton swab. The cells on the lower surface of the membrane were stained with Haematoxylin and Eosin. The invading cells were photographed under light microscope (Olympus BX41 laboratory microscope) fitted with a camera (Diagnostics instruments Inc.) at 20X magnification. Cells in each field were counted and plotted.

Apoptosis assay

Apoptosis was studied using the DeadEnd™ colorimetric TUNEL (TdT-mediated dUTP nick end labeling) system (Promega Corporation, WI). RF/6A cells (1 x 10 5 cells / well) were plated on cover slips in a 12-well plate and allowed to adhere for 24 hours. At the end 24 hours, the cells were exposed to different concentrations of Tf-T (1, 10, and

100 nM), tumstatin (100,250, and 500 nM), or bevacizumab (1, 10, and 100 nM) proteins. After 24 hours of protein exposure the cells were then washed, fixed with 4% paraformaldehyde, and permeabilized using 0.2% Triton® X-100 solution in phosphate buffer pH 7.4. The cells were then stained as per the manufacturer’s instructions. The cells were studied under a light microscope at 20X magnification.

Secretion of Tf-T fusion protein

RPE (retinal pigment epithelial) cells were used to evaluate the transport pattern of Tf-T protein. RPE cells (1 × 10 5 cells) were plated on Transwell filters (0.4µm pore size) and transepithelial electrical resistance (TEER) was measured using an EVOM® resistance meter (World Precision Instruments, CA). When TEER was above 200 .cm 2, tight junction formation was confirmed by ZO-1 staining. Filters with cells having a

TEER above 200 .cm 2 were fixed for 30 minutes at room temperature by addition of

10% formalin (0.5 ml on apical and 1.5 ml on basolateral side). Cells were further permeabilized by adding 0.1% triton X 100 containing 5% goat serum (0.5 ml on apical side) for 1 hour at room temperature. Primary ZO1 antibody, diluted to 1: 100 dilutions, was added to the cells and incubated for 1 hour at room temperature. Secondary FITC labeled antibody, diluted to 1: 100 dilutions, was added to cells and incubated for 1 hour at room temperature. Cells were incubated with 3 µg/ml DAPI (4',6-diamidino-2- phenylindole) for 5 minutes to stain the nuclei. Filters were then cut out with a sharp blade and transferred to a glass slide, fixed by adding SuperMount mounting media

(BioGenex, CA) and visualized under a confocal microscope.

To evaluate the secretion pattern, cells were incubated with 200 µg tumstatin or

Tf-Tor tumstatin protein. After 24 hours of transfection, the media was collected from

both the basolateral and the apical side separately and purified. Tumstatin and Tf-T proteins were quantified using a BCA® protein assay kit.

In silico protein modeling

An Accelery’s discovery visualizer v2.5.1.9167 (Accelry’s, Inc.CA) was used to study in silico docking of tumstatin and Tf-T to the αVβ3 integrin receptor. The crystal structure of the non-collagenous domain of collagen IV (PDB # 1LI1) was used as a reference to develop a homologous model for tumstatin. The crystal structure of iron-free human serum transferrin (PDB # 2HAV) was used as a reference to develop a homologous model for transferrin. The proteins were prepared and energy minimization was performed. Tf-T fusion protein was created by fusing tumstatin to the C-terminus of transferrin. For the tumstatin-αVβ3 integrin docking studies, the homology model of tumstatin was used as a ligand and docked onto the crystal structure of the extracellular domain of αVβ3 integrin (PDB # 1JV2). For the Tf-T-αVβ3 integrin docking studies, the fusion protein was docked to the αVβ3 integrin receptor (PDB # 1JV2).

Internalization of Tf-T protein

To study the internalization of Tf-T and tumstatin protein, 1 x 10 5 RF/6A cells / well were plated on cover slips in a 12-well plate and allowed to adhere for 24 hours.

RF/6A cells were exposed to 5 nM Tf-T-EGFP or tumstatin-EGFP protein for 24 hours.

After 24 hours the cells were washed with cold PBS pH 7.4 followed by washing with cold acidic buffer (pH 5.0) and fixed using 4% paraformaldehyde and stained with DAPI.

Cells were observed under a Nikon C1si® confocal microscope.

Efficacy studies in CNV induced Brown Norway rats

All animals were treated according to the Association for Research in Vision and

Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision

Research. The protocols for this study were approved by the Institutional Animal Care and Use Committee of the University of Colorado, Denver. Adult male Brown Norway

(BN) rats (150–180 gm) were purchased from Harlan Sprague Dawley Inc. (Indianapolis,

IN, USA). Rats were anesthetized using an intraperitoneal injection of 80 mg/ml ketamine and 12 mg/kg xylazine mixture.

Induction of laser burns was performed as published previously (Singh,

Grossniklaus et al. 2009; Tyagi, Kadam et al. 2012). Pupils were dilated by topical administration of 1% tropicamide solution. The fundus was visualized after instilling a drop of 2.5% hypromellose solution and placing a cover slip on the eye surface. Eight laser spots (100 mm, 150 mW, 100 ms) concentric with the optic nerve were placed in the right eye of each rat using a 532 nm diode laser (Oculight Glx; Iridex Inc., Mountain

View, CA, USA) and a slit lamp (Zeiss slit lamp 30SL; Carl Zeiss Meditec Inc., Dublin,

CA, USA). The left eye was used as a control for each animal. The Bruch’s membrane breakage was confirmed by the end point ‘bubble formation’. Rats showing intraocular hemorrhage or absence of bubble formation on laser administration were removed from the study. CNV lesions were allowed to develop for 14 days after induction of laser burns.

At the end of 14 days, rats were administered one of the treatments (a) PBS pH

7.4, (b) bevacizumab, or (c) Tf-T protein at a dose of 166 µM intravitreally. Intravitreal injections are commonly used to treat diseases such as CNV (Peyman, Lad et al. 2009).

The injection is performed under anesthesia. With the animal under anesthesia, cornea and sclera limbus was visualized and the conjunctiva and sclera was pierced with a 34G needle attached to a 10µl glass syringe 2 mm posterior to the limbus. In a similar set of experiments, 1-500 µM concentrations of Tf-T were given at the end of 14 days of CNV induction to assess a dose response.

At the end of 14 days after treatment, choroidal flatmounts were prepared as follows. The rats were anesthetized and infused with 10 ml of PBS (pH 7.4) followed by infusion with 10 ml 4% paraformaldehyde. Finally, 4 ml of 50 mg/ml fluorescein isothiocyanate (FITC)-dextran solution (2x10 6 Da) was infused. The eyes were then enucleated, fixed in 10% formalin for 24 hours, and choroidal flatmounts prepared as follows. Under a dissecting microscope, the anterior segment and crystalline lens were removed, and the retina was separated and removed from the eye with a fine scoop. In the remaining RPE-choroid-sclera, 4 radial cuts were placed and the tissue was ﬂattened. The sections were then ﬂatmounted on standard microscope slides, treated with mounting medium, and then further compressed with weights placed on top of the coverslips. After

24 hours, the flatmounts were imaged with a Nikon EZ-C1 confocal microscope using

488 and 568 nm excitation wavelengths. CNV areas were obtained using ImageJ software.

Results

Characterization of tumstatin and Tf-T protein

Figure 3.1 shows the characterization of tumstatin and Tf-T proteins. A single band at 100 kDa and 28 K Da in (Figure 3.1A) SDS PAGE and single peaks in (Figure

3.1C) SEC depicts the purity of Tf-T and tumstatin, respectively. Western blot of Tf-T

confirmed the protein (Figure 3.1B). Particle size for Tf-T and tumstatin was 8.0 nm and

3.9 nm, respectively (Figure 3.1D).

Figure 3.1: Characterization of proteins purified and used in the study. The (A) SDS- PAGE of Tf-T and tumstatin, (B) western blot of Tf-T using anti-transferrin antibody, (C) size exclusion chromatography (SEC) graph of Tf-T and tumstatin, and (D) particle size graph of Tf-T and tumstatin.

Inhibition of proliferation

Figure 3.2 shows the cell proliferation upon treatment with purified tumstatin, Tf-

T, and bevacizumab proteins. Tf-T was 19.8- and 1.6- fold superior to tumstatin and bevacizumab at inhibiting RF/6A cell proliferation. IC 50 for Tf-T, tumstatin, and bevacizumab was 8.3, 165.1, and 13.7 nM, respectively.

Figure 3.2: Improved anti-proliferative activity of Tf-T when compared to tumstatin and bevacizumab. MTT assay was used to assess the anti-proliferative activity of tumstatin,Tf-T, and bevacizumab on RF/6A cell proliferation stimulated by VEGF (50 ng/ml). Data is expressed as mean ± S.D. for 3 biological replicates.

Inhibition of endothelial tube formation

We then assessed the ability of our various proteins to inhibit endothelial organization via a tube formation assay. As shown in Figure 3.3, Tf-T was 11.3- fold superior to tumstatin protein at inhibiting tube formation, whereas no significant inhibition of tube formation was observed under the effect of bevacizumab. The IC 50 values for Tf-T and tumstatin were 3.5 and 39.75 nM, respectively. Figure 3.3B shows representative images of tube formation under the effect of the proteins at 250 nM.

Figure 3.3. Inhibition of tube formation observed in RF/6A cells. RF/6A cells were plated on Matrigel TM coated plates along with different concentrations of Tf-T, tumstatin, and bevacizumab protein. Tube formation was evaluated after 18 hours using light microscope at 10X magnification. (A) Plot for the inhibition of tube formation by tumstatin, Tf-T, and bevacizumab is shown. Data is expressed as mean ± S.D. for 3 biological replicates. (B) Representative images of RF/6A cell tube formation is shown at 250 nM.

Inhibition of invasion

The invasion assay makes use of a synthetic membrane which mimics the basement membrane in vivo, allowing cells to migrate towards a chemoattractant such as

VEGF. Figure 3.4A shows cell invasion observed in the presence of different

concentrations of Tf-T, tumstatin, and bevacizumab. Tf-T was 184.1- and 8.6- fold superior at inhibiting cell invasion than tumstatin and bevacizumab, respectively. The

IC 50 of Tf-T, tumstatin, and bevacizumab proteins were found to be 0.34, 62.6, and 2.95 nM, respectively. Figure 3.4B shows representative images of invading cells under the effect of 250 nM Tf-T, tumstatin, and bevacizumab.

Figure 3.4: Inhibition of endothelial cell invasion observed in RF/6A cells. (A) (A) Plot for the reduction in number of invading cells under the effect of Tf-T, tumstatin, and bevacizumab is shown. Data is expressed as mean ± S.D. for 3 biological replicates. (B) Representative images of RF/6A cell invasion is shown at 250 nM.

Induction of Apoptosis

The ability of Tf-T, tumstatin, and bevacizumab to induce apoptosis was then assessed by TUNEL assay. Figure 3.5 shows representative images of cells stained for

DNA ends in the presence of tumstatin, Tf-T and bevacizumab. Tumstatin and Tf-T protein induced apoptosis in RF/6A cells at all three concentrations tested (100, 250 and

500 nM and 1, 10 and 100 nM, respectively). No apoptosis was induced by bevacizumab

(1, 10 and 100 nM concentrations).

Figure 3.5: Tf-T and tumstatin induce apoptosis, whereas bevacizumab does not. Apoptosis induction by Tf-T and bevacizumab at concentrations of 1, 10, and 100 nM is shown. PBS was used as the control. Tumstatin was assessed at concentrations of 10, 250, and 500 nM. Each image is a representative image from three replicates. Arrows point towards apoptotic cell nuclei.

Basolateral secretion of Tf-T fusion protein

The optimal site of action for Tf-T is in the region lying between the RPE and the choroid where blood vessels are most prevalent. Cultured RPE cells formed tight junctions comparable to the endogenous tissue when grown on a Transwell filter for 4 weeks till TEER was above 200 .cm2 (Figure 3.6A). As a control, blank filters were also studied up to 4 weeks and TEER attained for the blank filters was subtracted from the TEER of the filters with cells growing on them (Figure 3.6A). Tight junction formation was also confirmed by the staining of ZO-1 protein. The RPE cells showed a clear staining pattern for ZO-1 and outlined the uniform polygonal shape of the RPE cells within the monolayer (Figure 3.6B). RPE cells secreted 126.8 µg/ml of Tf-T protein to the basolateral side (Figure 3.6C). On the contrary, only 12.3 µg/ml tumstatin was secreted towards the basolateral side.

In silico protein docking

Figure 3.7A shows the homology model for tumstatin that was developed using

Accelery’s discovery visualizer. Molecular models for (B) tumstatin and (C) Tf-T docked to the αVβ3 integrin receptor are also shown. The ZRANK score indicate that the docking of Tf-T (ZRANK score of -130.43) is more energetically favorable than the docking of tumstatin (ZRANK score of -113.28) alone. ZRANK score function combines electrostatics, van der Waals, and desolvation terms to calculate the energy of a protein- protein complex (Kastritis and Bonvin 2010).

Figure 3.6: Basolateral secretion of Tf-T protein in RPE cells grown on Transwell TM filters. (A) TEER values were measured up to ~ 200 .cm 2. Graph shows the TEER value over a period of 4 weeks. Blank filter TEER is shown as control. (B) Staining for expression of tight junction protein ZO-1 indicated the presence of tight junctions and hexagonal shapes of RPE cells. (C) Tf-T and tumstatin protein secreted towards the apical and basolateral side were quantified after incubating 200 µg protein with RPE cells for 24 hours. Data is represented as mean ± S.D. for n=4.

Figure 3.7: In silico modeling of tumstatin and Tf-T. Accelry’s discovery visualizer was used for in silico modeling. (A) Homology model of tumstatin protein developed is shown. Representative docked poses of tumstatin (B) and Tf-T (C) to αVβ3 integrin receptor are shown. The receptor is shown in red and tumstatin (B) or Tf-T (C) protein is shown in blue.

Figure 3.8: Tf-T-EGFP protein is internalized by RF/6A cells but not tumstatin-EGFP. A through C show GFP fluorescence, DAPI staining, and an overlay of RF/6A cells, respectively, exposed to 5 nM Tf-T-EGFP proteins. D through F show GFP fluorescence, DAPI staining, and an overlay of RF/6A cells, respectively, exposed to tumstatin-EGFP protein. 53

Internalization of Tf-T protein

Transferrin is rapidly internalized via the transferrin receptor. To determine if Tf-

T is also internalized we generated tumstatin-EGFP and Tf-T-EGFP fusion proteins as described in Materials and Methods. Cells were incubated with EGFP fusion proteins and subsequently washed thrice with cold PBS pH 7.4 followed by washing with cold acidic buffer (pH 5.0). Internalization of Tf-T-EGFP protein is shown in Figure 8. Intense EGFP signal is observed in RF/6A cells upon exposure to Tf-T-EGFP protein. Exposure of

RF/6A cells to the tumstatin-EGFP protein results in no internalization (Figure 3.8).

Efficacy studies in CNV induced Brown Norway rats

CNV was induced in rats via laser burns directed to Bruch’s membrane as described in Materials and Methods. After 14 days, rats were administered 166 µM doses of tumstatin, Tf-T, bevacizumab, or an equivalent volume of PBS by intravitreal injection. Tf-T was found to be 1.4 and 1.8 fold more efficacious in reducing laser induced CNV lesion area as compared to bevacizumab and tumstatin, respectively

(Figure 3.9A). A separate study of dose response assessment (Figure 3.9B) demonstrated that Tf-T had significant CNV reduction at 100 µM and above doses.

Discussion

We prepared a novel transferrin-tumstatin fusion protein and showed that 1) Tf-T inhibits endothelial cell proliferation with 19.8- and 1.6- fold greater potency than tumstatin and bevacizumab. 2) Tf-T was 184.1- and 8.6- fold superior at inhibiting cell invasion than tumstatin and bevacizumab, respectively. 3) Tf-T inhibits, tube formation with 11.3- fold higher potency than tumstatin, whereas bevacizumab doesn’t inhibit tube formation. 4) Tf-T is preferentially secreted towards the basolateral side across RPE cell

Figure 3.9: In vivo assessment of CNV lesion size in BN rats. Laser burns were induced in BN rats using a 532 nm diode laser. At the end of 14 days after laser induced burns, the rats were intravitreally treated with PBS pH 7.4, bevacizumab, tumstatin, or Tf-T. Rats were euthanized and eyes were enucleated at the end of 14 days of treatment. (A) A quantitative comparison of the treatments indicated superior efficacy of Tf-T as compared to bevacizumab and tumstatin. * indicates p<0.05 when compared to PBS and/or tumstatin treatment. † indicates p<0.05 when compared to bevacizumab. Data is expressed as mean ± S.D. for n=6. (B) Dose response curve using Tf-T at concentrations ranging from 1 to 500 nM. Data is expressed as mean ± S.D. for n=4.

monolayers. 5) Tf-T has significantly higher efficacy than tumstatin and bevacizumab in inhibiting choroidal neovascularization in a rat CNV model. Tf-T is predicted to have better binding interactions with αVβ3 integrin when compared to tumstatin.

Tf-T is superior in inhibiting cell proliferation in comparison to bevacizumab and tumstatin

Tf-T inhibited endothelial cell proliferation with 19.8- and 1.6- fold greater potency than tumstatin and bevacizumab, respectively (Table 3.1). We propose that the superior efficacy of Tf-T is the result of two possible mechanisms. One possible mechanism of improved activity of Tf-T could be resulting from the internalization of the fusion protein. We observed that Tf-T is internalized by RF/6A cells (Figure 3.8), whereas tumstatin is not. Endogenous antiangiogenic proteins (similar to tumstatin) such as endostatin and angiostatin have been shown to form complexes with RGD containing proteins such as fibronectin and vitronectin (Yi, Sakai et al. 2003). Accumulation of RGD containing proteins such as fibronectin in the cytoplasm of the cell can induce apoptosis

(Buckley, Pilling et al. 1999; Adderley and Fitzgerald 2000). Therefore, we speculate that

Tf-T increases the cytoplasmic content of RGD containing proteins such as fibronectin, thereby causing apoptosis of cells. This possibility is negligible for tumstatin as it did not show any signs of internalization in our studies. For tumstatin alone, its binding to αVβ3 integrin might be the principal mechanism of action.

Table 3.1: IC 50 values of tumstatin, Tf-T and bevacizumab in the three in vitro assays. N.D. indicates not determined.

Protein Inhibition of Inhibition of tube Inhibition of Proliferation (nM) formation (nM) invasion (nM)

Tumstatin 165.1 39.7 62.6

Tf-T 8.34 3.58 0.34

Bevacizumab 13.7 N.D. 2.95

Tf-T and tumstatin inhibit tube formation, whereas bevacizumab does not

Tf-T inhibits tube formation in RF/6A cells with 11.3- fold more efficacy than tumstatin alone, whereas, bevacizumab did not inhibit tube formation. In principle, tube formation occurs under the effect of collagen IV, VIII, XV, XVIII, and laminin 8 and 10, which are present in the basement membrane (Arnaoutova, George et al. 2009). These components tend to promote stability, adhesion, and invasion of endothelial cells. Growth factors such as VEGF are also present in the basement membrane matrix (Chen, Braet et al. 2002). However, VEGF does not seem to play an important role in tube formation, as tube formation occurs with high efficiency even in the presence of bevacizumab, which is known to act by neutralizing VEGF in the extracellular matrix. No inhibition of tube formation was observed at < 10 µg/ml bevacizumab in endothelial cells (Grau,

Thorsteinsdottir et al. 2011). It is perhaps not surprising that bevacizumab is unable to inhibit tube formation, given the array of non-VEGF drivers of this process.

However, in contrast to our results, reports are available wherein bevacizumab is shown to inhibit tube formation. This effect, however, requires large doses of bevacizumab.

Grau et al. (Grau, Thorsteinsdottir et al. 2011) reported that 100 µg/ml (~700nM) of

bevacizumab induced ~ 15% inhibition in tube formation. Xu et al. (Xu, Zhao et al. 2010) observed ~ 40% inhibition of tube formation using 2.5 mg/ml solution of bevacizumab.

In our study we observed that Tf-T inhibits tube formation at a much lower dose.

Tf-T is superior to tumstatin and bevacizumab in inhibiting RF/6A cell invasion

In the process of angiogenesis, endothelial cells invade the basement membrane.

We studied the invasion process using a Boyden chamber cell invasion assay. The purpose of this assay was to assess the impact of VEGF on invasion of basement membrane by endothelial cells that are further going to form blood vessels. Cell invasion under the effect of VEGF was significantly lower when Tf-T was used rather than when tumstatin was used alone. Tumstatin inhibits focal adhesion kinase (FAK) phosphorylation (Bakri, Snyder et al. 2007). Lack of FAK activation eventually leads to inhibition of cell invasion. Tf-T probably exerts similar effects, although to a greater extent. Bevacizumab, due to its ability to sequester VEGF, was effective at inhibiting invasion; however, Tf-T was superior to bevacizumab (IC 50 of 0.34 nM vs. 2.95 nM).

Tf-T and tumstatin induce apoptosis in RF/6A cells, whereas bevacizumab does not

Along with the limited success of the anti-VEGF therapies in treating CNV, there are inherent drawbacks in the anti-VEGF therapies too, including lack of apoptosis

(Kaempf, Johnen et al. 2008; Yoeruek, Tatar et al. 2010) in activated endothelial cells.

We observed greater anti-apoptotic activity for Tf-T followed by tumstatin. Tumstatin induces apoptosis by acting as a αVβ3 antagonist (Maeshima, Sudhakar et al. 2002). Tf-T inhibited cell proliferation, consistent with the ability of tumstatin to inhibit protein synthesis and apoptosis (Maeshima, Sudhakar et al. 2002). Bevacizumab did not exert

any apoptotic activity in RF/6A cells. Lack of apoptotic activity has been previously reported for bevacizumab in retina(Iandiev, Francke et al. 2011), corneal endothelial cells, retinal ganglion cells(Yoeruek, Tatar et al.; Kaempf, Johnen et al. 2008), and retina-

RPE-choroid cultures (Kaempf, Johnen et al. 2008). However, reports contrary to our results are also available, wherein bevacizumab has been shown to induce apoptosis in endothelial cells. Carneiro et al. (Carneiro, Falcao et al. 2009) showed that bevacizumab induced apoptosis in HUVEC cells at a concentration of 250 µg/ml and above. Similar to its effect on tube formation, bevacizumab is required in high doses to induce apoptosis.

For example, in comparison to results obtained by Carneiro et al,(Carneiro, Falcao et al.

2009) wherein ≥ 250 µg/ml showed apoptosis, Kaempf et al.(Kaempf, Johnen et al. 2008) observed no apoptosis in retina-RPE-choroid cultures at a bevacizumab concentration of

125 µg/ml. Furthermore, Tf-T requires much lower concentrations to induce apoptosis.

Tf-T protein is targeted towards the basolateral side of RPE cells

Another major advantage of fusing transferrin to tumstatin is the preferential basolateral secretion of Tf-T (Figure 3.6).Tf-T secretion towards the basolateral side of

RPE cell monolayer is 10-fold higher in comparison to tumstatin protein alone.

Basolateral localization endows Tf-T with the capability to target the proliferating choroid endothelial cells following intravitreal drug delivery. Intravitreal delivery of therapeutic agents has gained popularity and antibodies such as bevacizumab and ranibizumab are being administered intravitreally (Peyman, Lad et al. 2009). After intravitreal delivery of therapeutic agents, RPE acts as a barrier to the further movement of therapeutic agents. Tf-T is anticipated to be advantageous over tumstatin or bevacizumab by virtue of its vectorial transport from the apical to basolateral side of RPE

cells. Transport across RPE places Tf-T near choroid endothelial cells, the primary the site of disease.

Tf-T has superior in vivo anti-angiogenic efficacy when compared to tumstatin and bevacizumab

Our in vivo studies indicated the superior in vivo efficacy of Tf-T in inhibiting choroidal neovascularization. Lesion size in BN rat eyes treated with Tf-T was lower as compared to rats treated with bevacizumab or tumstatin. Tf-T was 1.4 and 1.8 fold superior than bevacizumab and tumstatin, respectively, in inhibiting CNV in a laser induced rat model when treatments were initiated at the end of day 14 of CNV induction

(Figure 3.9A). Our dose response curve (Figure 3.9B) demonstrated that a significantly superior effect is observed with a dose of ≥ 100 µM.

Conclusion

Together, our results suggest that Tf-T is a better therapeutic agent than tumstatin and bevacizumab because it has superior in vitro and in vivo efficacy in reducing CNV.

CHAPTER IV

PHARMACOKINETICS AND SAFETY OF A NOVEL TRANSFERRIN-

TUMSTATIN FUSION PROTEIN IN RABBITS

Abstract

Purpose

To determine the pharmacokinetics and safety of transferrin-tumstatin following intravitreal injection in rabbits.

Methods

Transferrin-tumstatin (Tf-T) was purified and conjugated to Alexa 488 dye. The alexa conjugated protein (250 µM) was injected intravitreally in New Zealand white rabbits (n=4) and monitored up to day 7 using non-invasive ocular fluorophotometry.

Pharmacokinetics was assessed using Winonlin. Safety of alexa conjugated Tf-T following intravitreal injection was assessed by ocular examination, wherein rabbits were monitored for chemosis, hyperemia, vitreal and aqueous humor floaters, and in intraocular pressure (IOP). Fundus imaging was used to assess the back of the eye for any changes. Clinical examination including change in body weight, temperature, heart rate was also performed. Blood was collected from marginal ear vein before and at the end of

7 days following intravitreal injection and evaluated for complete metabolic panel.

Results

Levels of alexa conjugated Tf-T decreased rapidly from 224.4 ng/ml to 160.5 ng/ml within the first three hours following injection and further decrease in levels was

slow and alexa conjugated Tf-T was observed in the vitreous up to day 7. Standard noncompartmental pharmacokinetic analysis determined the vitreal t 1/2 of Alexa conjugated Tf-T to be 1.7 days. Vitreal floater was observed in one rabbit eye at the end of 1 day of injection and disappeared at day 2 following injection. An increase in IOP was observed immediately upon injection, which returned back to normal within an hour.

Hyperemia at the site of injection was observed in 3 out of the 4 rabbits at day 1. Clincal parameters such as body weight showed no change during the study. Fundus imaging showed no change in the back of the eye. Complete metabolic evaluation of the serum demonstrated no change in the health of any organs.

Conclusions

Transferrin-tumstatin is retained in the vitreous for up to 7 days following a single intravitreal injection with a half life of 1.7 days. Transferrin-tumstatin was safe following single intravitreal injection in rabbits.

Introduction

Choroidal neovascularization is the leading cause for blindness in the USA.

Treatments currently available for CNV are based on blocking vascular endothelial growth factor (VEGF) or VEGF receptor. In 2004 and 2006, US Food and Drug

Administration (FDA) approved pegaptanib (Macugen, Eyetech), an aptamer that specifically blocks the VEGF165 isoform, and ranibizumab (Lucentis, Genentech Inc.), an anti-VEGF antibody that binds and inhibits all biologically active forms of VEGF-A, for the treatment of the neovascular (wet) AMD. Recently, using decoy VEGF receptor to prevent VEGF from binding to its receptors is described by many studies as the most effective method to block the VEGF signaling pathway (Ferrara, Hillan et al. 2004).

Therefore, this lead to the development of Eylea (Aflibercept, Regeneron, Inc.), which is a fusion protein of the second Ig domain of VEGF receptor 1 (VEGFR1), the third Ig domain of VEGF receptor 2 (VEGFR2), and the constant region (Fc) of human IgG1.

However, research has indicated that VEGF pathway blockade does not completely cure the pathogenesis of CNV (Rosenfeld, Brown et al. 2006). In a clinical trial with 716 patients, only 40% of the patients responded to ranibizumab treatment, with an improvement in visual acuity, with no patient being completely restored to 20/20 vision

(Soker, Takashima et al. 1998; Kompella, Sundaram et al. 2006; Rosenfeld, Brown et al.

2006; Ghate, Brooks et al. 2007; Hellstrom, Phng et al. 2007; Short 2008; Liang,

Butterfield et al. 2011; Kadam, Williams et al. 2013). Neuropilin 1(Soker, Takashima et al. 1998), jagged 1, and delta 4 (Hellstrom, Phng et al. 2007) are some of the proteins that have been recently discovered and shown to potentially mediate angiogenesis. Thus targets other than VEGF or its receptors are needed for efficient treatment of CNV.

We have developed a fusion protein transferrin-tumstatin that is based on tumstatin. Tumstatin is a collagen IV fragment and plays a pivotal role in inhibiting blood vessel formation by binding to its receptor, aVb3 integrin. Tumstatin has a distinct advantage over the existing treatments for CNV, as it induces apoptosis and is highly specific to angiogenic endothelial cell (because aVb3 integrins are primarily present on proliferating cells only). The capability to induce apoptosis is not present in ranibizumab and aflibercept because they merely act as VEGF scavenger. Furthermore, ranibizumab and aflibercept lack specificity as they don’t distinguish between VEGF needed for physiological growth and VEGF inducing angiogenesis. In addition to the promising prospect of tumstatin, transferrin, as a part of our fusion protein, can promote targeting

and enhanced uptake in cells (Kompella, Sundaram et al. 2006). Also, transferrin has been shown to have some anti-angiogenic property of its own and can augment the anti- angiogenic property of tumstatin (Liang, Butterfield et al. 2011).

The purpose of the present study was to isolate the pure Tf-T fusion protein and assess its safety and pharmacokinetics in rabbits. Rabbits are the most common species used for ocular safety and pharmacokinetic study because the rabbit eye is anatomically similar to humans and large enough to perform accurate ocular injections (Short 2008).

The fusion protein was injected intravitreally (IVT) into rabbits and ocular pharmacokinetics was examined using non-invasive ocular fluorophotometry. A specific sandwich ELISA was used to measure the intact transferrin-tumstatin fusion protein in rabbit plasma and ocular tissues.

Materials and methods

Materials

The 96 well half area plates (Costar Catalogue # 3690), secondary tumstatin antibody (C121068, LS Biosciences) and goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz

Biotechnology), Anti-transferrin antibody (D-9, sc-365871, Santa Cruz Biotechnology,

Inc,TX), TMB solution (Catalogue# 002023, Invitrogen Inc.)

Preparation and purification of Tf-T

Tumstatin and transferrin cDNA was PCR amplified and ligated into the vector

PSecTag2B using restriction enzyme and ligase method. ARPE cells (passage # 24) were grown in a T75 flask till 80 % confluency. Amplified Tf-T plasmid (cloned in

PSecTag2B vector) was diluted in DMEM/F12 media and mixed with lipofectamine reagent. The plasmid solution was transfected in ARPE cells and the cells were incubated

at 37°C in a CO 2 incubator for 24 hours. At the end of 24 hours, media was collected and purified using ion exchange chromatography. Ion exchange chromatography was carried out using AKTA FPLC (GE Healthcare life sciences, PA) fitted with a cationic SP sepharose bead filled column. Pure protein was eluted with increasing concentration of sodium chloride (0.2 to 1.0 M) in 25mM citrate phosphate buffer pH 6.0. Purified protein was further dialysed against 25mM citrate phosphate buffer pH 7.0 to remove excess of

NaCl. Protein purification was assessed using SDS PAGE. Purified protein was conjugated with Alexa 488 dye using Alexa Fluor® 488 Protein Labeling Kit. Endotoxin levels in the Alexa 488 conjugated Tf-T was tested using Pierce LAL chromogenic endotoxin quantitation kit.

Intravitreal injection in New Zealand white rabbits

Approval was obtained from the Institutional Animal Care and Use Committee at the University of Colorado, and the procedures adhered to the guidelines from the

Association for Research in Vision and Ophthalmology for animal use in research. Four

New Zealand white male rabbits weighing 1.7 to 2 kg (Harlan Laboratories, Indianapolis

IN) were anesthetized with an intramuscular injection of a mixture of ketamine (35 mg/kg) and xylazine (5 mg/kg). A 1% proparacaine hydrochloride ophthalmic drop was applied topically on the eye. The right eyes of the four New Zealand white rabbits were injected intravitreally with 50 µl of Alexa 488 conjugated Tf-T (250 µM solution) using a

30G needle (½ inch length) attached to a 100 µl Hamilton glass syringe. The left eyes were controls and received no intravitreal injections.

Monitoring of Alexa 488 conjugated Tf-T using ocular fluorophotometry

Ocular fluorophotometry was performed using Fluorotron Master (OcuMetrics,

Inc.CA) as described earlier (Ghate, Brooks et al. 2007; Kadam, Williams et al. 2013).

Attachment specific to the rabbits was used for measurement. Scans were collected at 5,

15, 30 minutes, 1, 2, 3, 4,6, 8, 10 ,12 hours, 1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5 and 7 days following intravitreal injections. Scans up to 30 minutes were performed while rabbit was under anesthesia. For other time points, rabbit was in restraint.

Tf-t vitreal concentration–time data were ﬁt by standard noncompartmental analysis to determine vitreal half-life, AUC, Cmax, and vitreous clearance using

WinNonlin (version 1.5, Pharsight, Mountain View, CA).

Assessment of safety parameters

Ocular examination: Chemosis and hyperemia was monitored to assess any effects at the site of injection. Regular images were taken using a phone camera. Clinical ophthalmology examination was performed using SL-D7 (Topcon Medical Systems, NJ) slit lamp system. Slit lamp was used to examine ocular inflammatory response such as anterior chamber cells and flare, and vitreal cells and floaters. Intraocular pressure (IOP) was measured using a Tono-Pen AVIA Applanation Tonometer (Reichert Technologies,

NY). The rabbit was held in a position wherein the Tono-Pen could be held perpendicular to the eye. Tono-Pen sensor was then tapped very lightly on the corneal surface. Each tap is recorded and a mean of 10 is provided per reading by the equipment. IOP measurement was performed at time points similar to ocular fluorophotometry. Fundus imaging was performed using Genesis Df fundus camera (Kowa Optimed Inc, CA). Lens attachment specific to rabbits was used in the fundus camera.Fundus images were used to look at the

fluorescence in the eye following injection and also to look at any damages following injection. Fundus camera was also used to capture images of any vitreal floaters. Fundus imaging was performed before injection, 15 minutes, 1, 2, 4, and 7 days after injection.

Clinical examination: Body weight (kg), heart rate (bpm), and temperature (°F) was assessed with the help of veterinarians in the animal housing facility before injection, and 1, 2, 4, and 7 days after injection. Blood collection was performed for comprehensive metabolic evaluation to assess any toxicity of the protein to other organs. Blood was withdrawn from the marginal ear vein using a 30G needle (½ inch length) attached to a

1ml disposable syringe (Exelint international, CA). Blood was withdrawn before injection, 1, 2, 4, and 7 days after injection and collected in BD vacutainer® blood collection tubes (BD Biosciences, NJ). Serum was obtained by allowing the blood sample to clot at room temperature for 1 hour, followed by centrifugation at 13000g for 15 minutes. The serum was frozen at 80° C until tested. For the blood metabolic parameter assessment, the serum was submitted to the clinical laboratory at the University of

Colorado hospital for the analysis of blood chemistry.

Eye enucleation and tissue separation: Rabbits were sacrificed at the end of 7 days using intravenous pentobarbital overdose (Fatal-Plus solution, Vortech

Pharmaceuticals, MI). Both eyes were immediately enucleated and were frozen in dry ice. The eyes were further frozen at 80° C until tested. For tissue separation, frozen eyes were cut at the limbus, and aqueous humor, and cornea separated. Following removal of cornea and aqueous humor, lens was scooped out of the eye. Frozen vitreous was then removed from the eye along with the retina. The remaining eye was defrosted and

choroid-retinal pigment epithelium (CRPE) was scrapped out. Sclera and conjunctiva were separated at the end from the defrosted eye.

Results

Preparation and purification of Tf-T

Purified Tf-T protein was confirmed using SDS-PAGE gel (Figure 4.1). Protein eluted using 0.2, 0.4, 0.7, and 1M sodium chloride (lane 7-10, Figure 4.1) was pooled together and dialysed against 25mM citrate phosphate buffer pH 7.0. Purified protein was conjugated to Alexa 488 dye and further assessed for endotoxin levels. The endotoxin level in the Tf-T was found to be below detection limit of 0.1 EU/ml and complies with the current US pharmacopoeial standard for sterile water for injection (0.25 EU/ml).

Figure 4.1: SDS PAGE gel of transferrin-tumstatin purified and used in the pharmacokinetic and safety study. Lane 1 is protein marker, lane 2 is control media with no Tf-T expression, lane 3 is media with Tf-T expression, lane 4 are 5 are flow through and wash from column. Lane 6-10 show Tf-T eluted with increasing concentration of NaCl.

Monitoring of Alexa 488 conjugated Tf-T using ocular fluorophotometry

Tf-T levels following intravitreal injection were monitored using ocular fluorophotometry (Figure 4.2). The peak for vitreous was identified based on the site of injection (Kadam, Williams et al. 2013). Levels of Alexa conjugated Tf-T decreased rapidly by ~30% from 224.4 ng/ml to 160.5 ng/ml within the first three hours following injection. Further decrease in levels of Alexa conjugated Tf-T was slow and Alexa conjugated Tf-T was observed in the vitreous up to day 7. Standard noncompartmental pharmacokinetic analysis determined the vitreal t 1/2 of Alexa conjugated Tf-T to be 1.7 days.

Figure 4.2: Intravitreal levels of alexa conjugated Tf-T equivalent to sodium fluorescein measured by ocular fluorophotometry. Data is represented as mean ± S.D. for n=4 rabbits. 69

Figure 4.3: Rabbit eye images captured before intravitreal injection, and 1 and 7 days after injection showing the appearance and disappearance of hyperemia (increased blood flow) at the site of injection. Images are representative for n=4 New Zealand white rabbits.

Figure 4.4: Fundus images showing vitreal floater observed in 1 out of 4 New Zealand white rabbits following intravitreal administration of Tf-T. Arrow points to the floater after 1 day of injection, which disappeared two days following injection.

Figure 4.5: Intraocular pressure in rabbit eyes dosed with Tf-T. Contralateral eye is the undosed eye of the rabbits and serve as a control. Data is represented as mean ± S.D. for n=4 rabbits.

Figure 4.6: Angiography and bright field images rabbit eyes dosed with Tf-T. Data is represented as mean ± S.D. for n=4 rabbits.

Assessment of safety parameters

Ocular examination: An increase blood flow (hyperemia) was observed at the site of injection in 3 out of the 4 rabbits tested. Figure 4.3 shows the site of injection with the enhanced blood flow (marked by a circle and arrow). The increased blood flow disappeared in all the animals by the end of day 7 following injection. A vitreal floater

(Figure 4.4) was observed in one rabbit eye out of the four rabbits tested at the end of 1 day of injection. The floater disappeared at day 2 following injection and was not observed at any further time point.

Intraocular pressure (IOP) (Figure 4.5) of the dosed eye increased from 10.7 ± 1.7 mmHg to 29.7± 7.4 mmHg upon injection. However, the IOP returned back to 11 mmHg at the end of 1 hour. No change in IOP was observed in the contralateral undosed eye.

The IOP in the contralateral undosed eye was 10.5 ± 0.8 mmHg during the entire study period. Fundus imaging demonstrated the presence of fluorescence in the eye following intravitreal injection (angiography in Figure 4.6). Furthermore, bright field fundus images showed lack of toxicity to the back of the eye (Figure 4.6).

Clincal examination: No changes were observed in the body weight, heart rate, and temperature over the period of 7 days following treatment. Details are provided in

Table 4.1. Table 4.2 gives the results of the comprehensive metabolic evaluation performed on rabbit serum collected before initiation of study and at the end of 7 days following intravitreal injection of Tf-T. Values of all the parameters assessed were withint the acceptable range.

Table 4.1: Results of clinical examination performed on New Zealand white rabbits before intravitreal injection, and 1 and 7 days after injection of Tf-t. Data is represented as mean ± S.D. for n=3 rabbits.

Parameter Before injection Day 1 Day 7

Body Weight (kg) 2.52 (± 0.11) 2.53(± 0.11) 2.57(± 0.09)

Heart rate (bpm) 202.33(± 26.57) 222.67(± 24.11) 230(± 42.42)

Temperature (F) 100.67(± 1.09) 102.63(± 1.73) 101.20(± 0.84)

Table 4.2: Results of comprehensive metabolic evaluation performed on New Zealand white rabbits before intravitreal injection, and at the end of 7 days after injection of Tf-T. Data is represented as mean ± S.D. for n=3 rabbits.

Chemistry Units Mean (± S.D.) Acceptable range Before treatment 7 days after single dose of Tf-T Sodium mmol/L 142.0 (± 9.5) 144.3 (± 7.0) 133-145 Potassium mmol/L 4.6 (± 0.4) 4.5 (± 0.7) 3.3-5.0 Chloride mmol/L 104.3 (± 4.7) 107.7 (± 2.9) 101-111 CO 2 mmol/L 24.0 (± 4.6) 21.7 (± 1.5) 22-32 Glucose mg/dL 124.7 (± 6.7) 129.7 (± 2.9) 70-199 Blood urea mg/dL 12.7 (± 0.6) 12.0 (± 1.0) 6 - 23 nitrogen Creatinine mg/dL 1.0 (± 0.1) 0.9 (± 0.1) 0.4-1.2 Calcium mg/dL 10.4 (± 1.8) 10.0 (± 1.3) 8.5-10.3 Total Protein g/dL 7.2 (± 0.8) 7.1 (± 0.7) 6.4-8.3 Albumin g/dL 1.8 (± 0.1) 1.8 (± 0.1) 3.4-5.0 Bilirubin mg/dL 0.7 (± 0.5) 1.1 (± 0.8) 0.0-1.3 AST U/L 46.0 (± 3.6) 36.7 (± 2.5) 0-47 ALT U/L 21.3 (± 4.2) 23.3 (± 8.5) 0-47 ALK P U/L 101.0 (± 10.1) 98.0 (± 12.2) 39-117

Discussion

In this study we have concluded that (1) alexa conjugated Tf-T is cleared from the vitreous humor following intravitreal injection in 1.7 days, (2) intravitreal injection of Tf-

T induces temporary hyperemia at the point of entry of needle, (3) intravitreal injection of

Tf-T causes a very short termed increase in IOP, and (4) Tf-T has no effect on the functioning of other bodily organs.

Alexa conjugated Tf-T had a half life of 1.7 days in the vitreous. Fusion proteins similar to Tf-T have been reported to have a longer half life. Aflibercept, which is a 100

KDa, intravitreally injected fusion protein has a vitreal half life of 4.7 days in humans

(Stewart and Rosenfeld 2008). The difference in vitreal half lifes of Tf-T and a similar protein, aflibercept, can be due to the difference in species wherein the half life was evaluated (rabbits for Tf-T vs. humans for aflibercept). Proteins have been reported to diffuse into the anterior chamber, where they exit through the aqueous outflow system

(Bakri, Snyder et al. 2007; Krohne, Eter et al. 2008). As the rate of intraocular drug loss is partly controlled by diffusion (Maurice and Mishima 1984) larger eyes with longer diffusion vitreous paths would be expected to have slower vitreous clearance. As human eyes have a larger vitreous volume (~ 5 ml) (Oyster 1999) in comparison to rabbits (~1.5 ml) clearance of proteins is also expected to be faster from rabbit vitreous.

Temporary side effects of intravitreal injection such as conjunctival hyperemia at the site of entry of needle in to the vitreous and vitreal floaters were observed in some of the rabbits evaluated in our study. Hyperemia was observed in 3 out of the 4 rabbits evaluated. However, the hyperemia was temporary and cleared by the end of the study.

Hyperemia is one of the most common side effects observed in the intravitreal treatments and is most likely the result of the anesthesia technique rather than the injection procedure itself (Costa, Jorge et al. 2006; Georgopoulos, Polak et al. 2009). Another temporary adverse effect was vitreal floaters following intravitreal delivery of Tf-T.

Vitreal floaters are considered a common but not serious adverse effect. In our study the floater appeared on day 1 following injection in one rabbit and was a dark circular spot.

The floater disappeared by day 2 and was not observed any time further. The floater could have an air bubble which was later abosorbed.

In addition to hyperemia and a vitreal floater, other complications of intravitreal injection of Tf-T included increase in IOP. Short term increase in IOP has been reported following injection of bevacizumab (Hollands, Wong et al. 2007) and ranibizumab(Rosenfeld, Brown et al. 2006). However, there is no agreement amongst researchers regarding the occurrence of IOP rise, with publications reporting lack of rise in IOP (Spaide, Laud et al. 2006). Probable reasons for spike in IOP are the volume injected into the vitreous(Rosenfeld, Brown et al. 2006), the needle bore size employed for the injection (Kim, Mantravadi et al. 2008), and sclera rigidity.

Conclusion

Tf-T has no serious side effects and is well tolerated following a single intravitreal injection.

CHAPTER V

COMPARISON OF SUPRACHOROIDAL DRUG DELIVERY WITH

SUBCONJUNCTIVAL AND INTRAVITREAL ROUTES USING NONINVASIVE

FLUOROPHOTOMETRY 1

Abstract

Purpose

To determine whether exposure of sodium fluorescein (NaF) to the choroid-retina region in the posterior segment of the eye is greater with suprachoroidal injection when compared to intravitreal and transscleral routes.

Methods

Suprachoroidal injection, a new approach for drug delivery to the posterior segment of the eye was validated using a 34 G needle and Indian ink injections in

Sprague Dawley rats, followed by histology. Delivery of NaF was compared in Sprague

Dawley rats after suprachoroidal, posterior subconjunctival, or intravitreal injections.

NaF levels were monitored noninvasively up to 6 hours using Fluorotron Master™, an ocular fluorophotometer Pharmacokinetic parameters were estimated using WinNonlin.

1 Puneet Tyagi, Rajendra S. Kadam, Uday B. Kompella (2012) Comparison of Suprachoroidal Drug Delivery with Subconjunctival and Intravitreal Routes Using Noninvasive Fluorophotometry, PLoS ONE: e48188. doi:10.1371/journal.pone.0048188

Results

Histological analysis indicated localization of India ink to the suprachoroidal space below sclera, following injection. NaF delivery to choroid-retina was in the order: suprachoroidal > intravitreal > posterior subconjunctival injection. Peak NaF concentration (Cmax) in choroid-retina was 36-fold (p=0.001) and 25-fold (p=0.001) higher after suprachoroidal (2744 ± 1111 ng/ml) injection when compared to posterior subconjunctival (76 ± 6 ng/ml) and intravitreal (108 ± 39 ng/ml) injections, respectively.

NaF exposure (AUC0-360min) to choroid-retina after suprachoroidal injection was 6-fold

(p=0.001) and 2-fold (p= 0.03) higher than posterior subconjunctival and intravitreal injections, respectively. Choroid-retina Tmax was observed immediately after dosing with suprachoroidal injections and at 10 and 27.5 minutes, respectively, with subconjunctival and intravitreal injections.

Conclusions

Suprachoroidal injections are feasible in a rat model. Suprachoroidal injections resulted in the highest bioavailability, that is, the extent and rate of delivery of NaF to choroid-retina, when compared to intravitreal and posterior subconjunctival injections.

Ocular fluorophotometry is useful for noninvasive monitoring of NaF in rats following administration by various routes including suprachoroidal route.

Introduction

Diseases of the posterior segment of the eye are responsible for severe vision loss and blindness in the developed countries. The most prevalent posterior segment diseases include age related macular degeneration (AMD), diabetic retinopathy, and retinal degenerative diseases. As of 2008, AMD is prevalent in 8 million in the USA and is

expected to increase to 12 million by 2020 (Jager, Mieler et al. 2008). Nearly 10% of the subjects suffering from AMD are diagnosed with the growth of abnormal or leaky blood vessels in the choroid below the retina, a condition known as wet AMD or choroidal neovascularization (CNV). CNV is primarily responsible for significant loss of vision and blindness in AMD patients. Diabetic retinopathy is prevalent in 4.1 million people in the

United States, with nearly 22% (0.9 million) of diabetic patients having vision threatening diabetic retinopathy (Saaddine, Honeycutt et al. 2008). Further, the number of diabetic patients in the USA is expected to rise to 16 million by 2050 (Saaddine,

Honeycutt et al. 2008). Increase in prevalence of these vision threatening disorders is also resulting in a rise in the cost of treatment (Smiddy 2007). Despite the severity and increasing prevalence of back of the eye diseases, conventional drug delivery methods are either inefficient in delivering required amount of drug to the site of action or highly invasive to the vitreous humor, with significant side effects.

The most common drug delivery method for treating ocular disorders is topical administration, primarily due to its convenience. Unfortunately, topically administered treatments are rapidly drained from the ocular surface, resulting in less than 5% bioavailability, that too mainly to the tissues in the anterior segment of the eye (Lee and

Robinson 1986). Due to the barriers present, currently there is no eye drop formulation approved for treating back of the eye diseases. To bypass the barriers associated with topical delivery for back of the eye diseases, intravitreal injections are becoming popular

(Anderson, Bainbridge et al. 2010; Shelke, Kadam et al. 2011). However, intravitreal injections are highly invasive and associated with complications such as cataract, retinal detachment, vitreous hemorrhage, and endophthalmitis (Jonas, Spandau et al. 2008).

Other than topical and intravitreal routes of delivery, periocular routes such as sub-Tenon and subconjunctival routes can also be used to deliver drugs to the posterior segment of the eye (Amrite, Ayalasomayajula et al. 2006; Amrite, Edelhauser et al. 2008). The periocular routes place the therapeutic agent adjacent to the sclera for transscleral delivery, thereby reducing the risks associated with the intravitreal route of administration (Raghava, Hammond et al. 2004). Nevertheless, periocular routes have disadvantages such as hemorrhage at the site of injection (Cheruvu, Ayalasomayajula et al. 2003; Negi, Browning et al. 2006). Thus, development of a safe and efficacious route of delivery for the treatment of posterior segment disorders remains the foremost challenge in ocular drug delivery research.

Suprachoroidal space (SCS) (Krohn and Bertelsen 1997) is a unique, anatomically advantageous space that localizes therapeutic agents adjacent to the choroid-retina region, the target tissue affected in the neovacular form of age related macular degeneration and diabetic retinopathy. Safety of injections into the SCS was shown by Einmahl et al.(Einmahl, Savoldelli et al. 2002), wherein a novel poly (ortho ester) biomaterial was evaluated, and by Poole et al.,(Poole and Sudarsky 1986) wherein sodium hyaluronate was injected to treat retinal detachments. Einmahl et al.,(Einmahl, Savoldelli et al. 2002) observed that poly (ortho ester) injection in the SCS caused no clinical complications except some slight choroidal pigmentation and presence of vacuoles in the SCS. Poole et al., (Poole and Sudarsky 1986) observed slight bleeding and inflammation at the site of injection, which disappeared within 3 weeks. Olsen et al. (Olsen, Feng et al. 2006) evaluated the safety of a novel cannula system for delivery in the SCS by monitoring histopathology and retinal and choroidal blood flow in monkeys and pigs and observed

minor tissue injury at the site of injection. More recently, Patel et al.(Patel, Lin et al.

2011) developed and evaluated a minimally invasive strategy using a novel hollow microneedle system to study the ex vivo suprachoroidal distribution of sulforhodamine B dye and particles ranging in size from 20 to 1000 nm. Suprachoroidal delivery is minimally invasive and might be safer because it does not require entry into the vitreous, thereby potentially protecting retina from any injection related damage.

Even though suprachoroidal delivery is being evaluated for effective treatment of posterior segment disorders, there are no reports comparing it to periocular injections.

Further, there are limited investigations comparing suprachoroidal and intravitreal routes of delivery, that too for a protein drug but not small molecules (Olsen, Feng et al. 2011).

Since choroid vessels have high blood flow, it is generally perceived that drug molecules can clear very rapidly. Therefore, a direct comparison of different routes of drug administration will help establish the relative advantage of suprachoroidal delivery.

We used a non-invasive ocular fluorophotometry technique to study the distribution of NaF following different routes of injection. Following periocular injections, a few pharmacokinetics studies have been conducted using ocular fluorophotometry for small molecules such as NaF (Ghate, Brooks et al. 2007) and oregon green–labeled triamcinolone acetonide (Lee, Kim et al. 2008) and macromolecules such as high molecular weight FITC-dextran (40 kDa and 70kDa)

(Berezovsky, Patel et al. 2011). Traditional methods of evaluating ocular pharmacokinetics are invasive and costly. Sacrificing animals at multiple time points followed by eye enucleation and isolation of different ocular tissues makes the process tedious and time consuming. Further, changes in drug location and concentration can

occur during tissue extraction. In comparison, ocular fluorophotometry is a non-invasive technique, which does not affect ocular tissues and allows time course evaluations in the same animal in different ocular tissues using a single scan. In this study, we determined the delivery and pharmacokinetics of NaF injected in suprachorodial space of rats and compared it with intravitreal and posterior subconjunctival injections using ocular fluorophotometry. NaF is a rational choice for in vivo fluorophotometry because of its safety (Das and Vedantham 2004), high absorptivity, and fluorescence yield (Sjoback,

Nygren et al. 1995). Further, the molecular weight of NaF (376 Da) is similar to many antimicrobial agents and steroids administered to the eye for the treatment of ocular disorders.

Materials and methods

Materials

Sodium fluorescein (NaF) used in this study was purchased from Sigma-Aldrich

(St. Louis, MO).

Ethics statement

All animals were treated according to the Association for Research in Vision and

Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision

Research. Animal protocols followed during this study were approved by the Institutional

Animal Care and Use Committee of the University of Colorado Anschutz Medical

Campus, Aurora, CO.

Administration of NaF by different routes

Adult male Sprague Dawley (SD) rats (150–180g) were purchased from Harlan

Sprague Dawley Inc. (Indianapolis, IN, USA). Rats were anesthetized using an

intraperitoneal injection of a mixture of 80 mg/kg ketamine and 10 mg/kg xylazine.

Using a 10µl Hamilton glass syringe (Hamilton company, NV) fitted with a 34 gauge needle (1/2 inch long; with a 45 o taper), 5 µl phosphate buffer saline (PBS; pH 7.4) containing 100 µg/ml NaF was injected into the suprachoroidal space, posterior subconjunctival region, or vitreous humor of the right eye of the rat.

Histology of rat eye after suprachoroidal injection

To confirm the accuracy of suprachoroidal injection in vivo in rat eyes, histological examination of rat eye after suprachoroidal injection of India ink dispersion was performed. Rats were anaesthetized and 5µl of 5% India ink dispersion was injected in the suprachoroidal space using a 10µl Hamilton glass syringe fitted with a 34 gauge needle (1/2 inch long; with a 45 o taper). Rats were immediately euthanized and eyes enucleated. Eyes were further fixed in 4% formalin solution for 2 days. Paraffin sections

(5 um thick) were obtained and stained with haematoxylin and eosin. Sections were observed under a light microscope (Olympus BX41 laboratory microscope) fitted with a camera (Diagnostics instruments Inc.).

Ocular fluorophotometry

The disposition of NaF was studied using Fluorotron Master TM , an ocular fluorophotometer (OcuMetrics Inc., Mountain View, CA) fitted with a small animal adapter. The Fluorotron scans report NaF concentrations in ocular tissues at 0.25 mm intervals along an optical axis used by the instrument. Prior to acquiring Fluorotron scans, a single drop of 1% tropicamide (Mydriacyl, Alcon laboratories, Inc., TX) solution was instilled in the eyes. Baseline fluorescence was measured prior to NaF injections.

After NaF injections, Fluorotron scans were acquired up to six hours, with repeated

application of tropicamide drops every 2 hours. The maximum pupil diameter size

(pupillary dilation) with 1% tropicamide solution eye drops is attained within ~ 40 minutes (Park, Lee et al. 2009) and persists up to ~ 70 minutes, requiring repeated instillation. Saline solution eye drops were applied to the eyes periodically, to prevent dehydration of the corneas. The raw data from the fluorophotometer was transferred to a spreadsheet (Excel; Microsoft Corporation, WA) and plotted.

Pharmacokinetic and statistical analysis

Non-compartmental pharmacokinetic analysis for the three routes of injection was performed using WinNonlin software (Version 1.5, Scientific Consulting, Inc.). AUC 0-360 min is the area under the curve obtained by plotting the concentration-time data, where‘t’ is the last time point at which NaF levels were measured. The “t” value was 360 minutes for the three routes of administration. The 0-time point concentration was considered as zero when the drug was measured away from the site of dosing (extravascular dose mode in WinNonlin). When the drug was measured at the site of administration (e.g., estimation of choroid levels after suprachoroidal injection or vitreal levels after intravitreal injection), WinNonlin estimated the 0-time concentration by extrapolating the data to y-axis. A statistical comparison of the pharmacokinetic parameters was performed using one-way ANOVA followed by Tukey's post hoc analysis (SPSS, ver.11.5; SPSS,

Chicago, IL). The results were considered statistically significant at p < 0.05.

Results

Histology of rat eye after suprachoroidal injection

Since this was the first study to evaluate the pharmacokinetics of NaF after suprachoroidal injection in rats, the accuracy of the suprachoroidal injection was

confirmed by histological sectioning of India ink injected SD rat eyes (Figure 5.1). The histological cross section of India ink injected SD rat eyes showed a spread of India ink between the sclera and choroid. Suprachoroidal injection resulted in widening of suprachoroidal space as compared to control eyes (Figure 1D), which might be due to the pressure created by the India ink injection. Similar widening of suprachoroidal space was also observed by Patel et al. (Patel, Lin et al. 2011). SD rat eyes without any injection of

India ink were used as the negative control, which showed no black color in any part of the eye (Figures 5.1A and 5.1C).

Fluorophotometric measurement

The Fluorotron Master is calibrated to provide readouts of fluorescence in NaF concentrations. Thus, readings from the scans were directly used as NaF concentrations in a given region of the eye. In the Fluorotron Master a blue excitation light is delivered through the optics of the system to the eye and the resulting emitted fluorescent light is collected via the same optical system. A measurement area is created at the point where the excitation and emission lights intersect and is known as the focal diamond (Raines

1988). The focal diamond, a measure of resolution inside the rat eye, is 400 µm. Levels of fluorescence are measured within this focal diamond, and the focal diamond is automatically moved along the axis of the eye in the posterior to anterior direction.

Following the above protocol, we obtained scans for blank eyes and eyes injected with NaF by different routes. NaF concentrations in the eye were plotted against distance data points separated by 0.25 mm on an optical axis. This distance in millimeters on the plot cannot be related to the actual dimensions of rat eye tissues. However, potential tissue assignments to data points can be made based on tissue autofluorescence peaks and

by monitoring fluorescence signal following sodium fluorescein injections in various compartments of the eye. Figure 5.2 shows representative Fluorotron scans for blank eyes

(Figure 5.2A) and eyes injected with NaF by suprachoroidal, posterior subconjunctival, and intravitreal routes immediately (Figures 5.2B) and 30 minutes after injection (Figure

5.2C).

Based on the known ability of choroid, lens, and cornea to autofluoresce, data points at approximately 25, 61, and 88 in baseline Fluorotron scans were considered as autofluorescence peaks corresponding to choroid, lens, and cornea, respectively.

Following NaF injections, peak signals for posterior subconjunctival, suprachoroidal, and intravitreal injections were evident at data points 10, 21, and 45, respectively, in the

Fluorotron scans (Figure 5.2D-E). The vitreal peak at 45 th data point (Figure 5.2F) was also observed by Ishiko et al. during a fluorescein distribution study in a tree shrew

(Ishiko, Yoshida et al. 1996). The blank eye scans (Figure 5.2A) (n = 6) showed 6.5 (±

Therefore, NaF concentrations in dosed animals at data points 21, 45, and 77 were assigned as concentrations in choroid-retina, vitreous, and, anterior chamber respectively, for pharmacokinetic analysis.

Figure 5.1: Suprachoroidal injection of India ink between sclera and choroid-retina in SD rats. Eyes were injected with 5 µl of 5 % India ink dispersion in suprachoroidal space. The eyes were fixed in 4% formalin and embedded in paraffin blocks. H & E stained sections were examined. (A) A 4x magnification image showing a cross section of a blank eye; (B) Eye administered with suprachoroidal injection; (C) 10x magnification of a blank eye; and (D) A 10x magnification of the site of suprachoroidal injection showing the presence of India ink in the suprachoroidal space.

Figure 5.2: Representative fluorophotometry scans attained using Fluorotron Master™ in Sprague Dawley rat eye. Scans are for (A) blank eye showing autofluorescence, (B) eyes immediately after injection of NaF in suprachoroidal, posterior subconjunctival, or vitreous region, (C) eyes 30 minutes after injection of NaF in suprachoroidal, posterior subconjunctival, or vitreous region. Data in panel A is an average for n=6, and in B and C it is an average for n=4. Representative time dependent scans after injection of NaF in (D) suprachoroidal, (E) posterior subconjunctival, and (F) vitreous regions are also shown. Blank eye scan shows the autofluorescence of choroid-retina, lens, and cornea regions.

Following NaF injections, peak signals for posterior subconjunctival, suprachoroidal, and intravitreal injections were evident at data points 10, 21, and 45, respectively, in the

Fluorotron scans (Figure 5.2D-E). The vitreal peak at 45 th data point (Figure 5.2F) was also observed by Ishiko et al. during a fluorescein distribution study in a tree shrew

(Ishiko, Yoshida et al. 1996). The blank eye scans (Figure 5.2A) (n = 6) showed 6.5 (±

5.53), 12.3 (± 7.5), and 15.7 (± 3.2) ng/ml autofluorescence measured as sodium fluorescein equivalents in choroid, lens, and cornea regions, respectively. The autofluorescence for the anterior chamber in the valley between lens and cornea peaks at data point 77 was 0.5 ± 0.31 ng/ml (n = 6). Therefore, NaF concentrations in dosed animals at data points 21, 45, and 77 were assigned as concentrations in choroid-retina, vitreous, and, anterior chamber respectively, for pharmacokinetic analysis.

NaF pharmacokinetics after suprachoroidal injection

Figure 5.3 shows the mean NaF concentration in different regions of the eye after suprachoroidal injection of NaF. The amount of fluorescein in the choroid-retina region observed immediately after the injection was 1673 ± 363 ng/ml (at 2 minutes). The concentrations extrapolated to time zero by WinNonlin were 2744 ± 1111 ng/ml. The peak fluorescein concentrations in the vitreous and the anterior chamber regions were 597

± 297 and 122 ± 34 and ng/ml, and 3- and13- fold lower, respectively, than the choroid- retina region. The concentration of fluorescein peaked at 10 minutes in all animals in the vitreous as well as the anterior segment regions.

Figure 5.3: Sodium fluorescein concentrations in choroid-retina, vitreous, and anterior chamber regions after injection in the suprachoroidal space. At 2 minutes after the injection, the choroid-retina had the highest concentration (1673 ± 363 ng/ml). Vitreous and anterior chamber regions had peak concentrations of 597 ± 297 ng/ml and 122 ± 34 ng/ml, respectively, at 10 minutes. The baseline values for choroid, vitreous, and anterior chamber were 6.5 (± 5.53), 1.65 (± 0.78), 0.5 (± 0.31) ng/ml, respectively. Data is presented as mean ± S.D. for n=4.

NaF pharmacokinetics after subconjunctival injection

Mean NaF concentrations in choroid-retina, vitreous, and anterior segment regions after posterior subconjunctival injection are shown in Figure 5.4. The peak concentrations in the choroid-retina and vitreous regions observed at 10 ± 0 and 67.5 ±

115 minutes (3 animals had peak at 10 minutes and one exhibited a peak at 240 minutes) were 76 ± 6 and 17 ± 13 ng/ml, respectively. Anterior chamber region showed peak fluorescein value of 11 ± 1 ng/ml at 30 ± 0 minutes.

Figure 5.4: Sodium fluorescein concentration in choroid-retina, vitreous, and anterior chamber regions after posterior subconjunctival injection. At 10 and 67.5 minutes after injection, the choroid-retina and vitreous regions had the highest concentrations of 76 ± 6 and 17 ± 13 ng/ml, respectively. Anterior chamber region had a peak concentration of 11 ± 1 ng/ml at 30 minutes. The baseline values for choroid, vitreous, and anterior chamber were 6.5 (± 5.53), 1.65 (± 0.78), 0.5 (± 0.31) ng/ml, respectively. Data is presented as mean± S.D. for n=4.

NaF pharmacokinetics after intravitreal injection

Figure 5.5 shows the mean values for NaF concentration in choroid-retina, vitreous, and anterior chamber regions after intravitreal injection of NaF, with the peak vitreous concentrations measured at 2 minutes being 1512 ± 1517 ng/ml. The concentrations extrapolated to time zero were 2004 ± 2268 ng/ml. The highest NaF concentration (103 ± 44 ng/ml) in the choroid-retina region was observed at 27.5 ± 23.6 minutes and it was 15- fold lower compared to the peak values observed in the vitreous.

The anterior chamber region had a peak concentration of 24 ± 8 ng/ml at 10 ± 0 minutes. 91

Figure 5.5: Sodium fluorescein concentrations in choroid-retina, vitreous and anterior chamber regions after intravitreal injection. At 2 minutes of injection, the vitreous region had the highest concentration (1512 ± 1517 ng/ml). Choroid-retina and anterior chamber regions had peak concentrations of 103 ± 45 ng/ml and 24 ± 8 ng/ml, respectively, at 27.5 minutes and 10 minutes. The baseline values for choroid, vitreous, and anterior chamber were 6.5 (± 5.53), 1.65 (± 0.78), 0.5 (± 0.31) ng/ml, respectively. Data is represented as mean ± S.D. for n=4. Inset shows NaF levels in choroid-retina and anterior chamber.

Comparison of pharmacokinetic parameters and drug concentrations between different routes for choroid-retina delivery

Cmax and AUC 0-360 min data is shown in Figure 5.6. The area under curve (AUC 0-360 min ) for NaF concentration in the choroid-retina region was 6-fold (p=0.001) and 2-fold

(p= 0.03) higher than posterior subconjunctival and intravitreal injections, respectively.

The C max of choroid-retina region was 36-fold (p=0.001) and 25-fold (p=0.001) higher after suprachoroidal injection compared to subconjunctival and intravitreal injections, respectively. The time to attain maximum concentration (T max ) in choroid-retina region was in the order: suprachoroidal injection < subconjunctival injection < intravitreal injection. T max for vitreous region was in the order: intravitreal injection < suprachoroidal injection < subconjunctival injection. T max for anterior chamber was 10 minutes for suprachoroidal and intravitreal routes and 30 minutes for subconjunctival route.

At 2 minutes, choroid-retina levels were significantly higher (p<0.05) after suprachoroidal injection when compared to intravitreal and subconjunctival injections. At

30 minutes, choroid-retina levels were significantly higher (p<0.05) after suprachoroidal injection when compared to subconjunctival injection. At 60 minutes, choroid-retina levels were significantly higher (p<0.05) after intravitreal injection when compared to subconjunctival injection. At 120 minutes, choroid-retina levels were significantly higher

(p<0.05) after intravitreal injection when compared to suprachoroidal and subconjunctival injections.

At 10, 60, 120, and 240 minutes, vitreous levels were significantly higher

(p<0.05) after intravitreal injection when compared to subconjunctival and

suprachoroidal injections. At 10 and 30 minutes, vitreous levels were significantly higher

(p<0.05) after suprachoroidal injection when compared to subconjunctival injection.

At 2, 30, and 60 minutes, anterior chamber levels were significantly higher (p<0.05) after suprachoroidal injection when compared to subconjunctival injection. Anterior chamber concentrations were significantly higher (p<0.05) after intravitreal injection when compared to subconjunctival injection at 2, 10, 30, and, 60 minutes.

Figure 5.6: Pharmacokinetic parameters (C max and AUC 0-360min ) estimated for sodium fluorescein after injection by suprachoroidal, intravitreal, and posterior subconjunctival routes in Sprague Dawley rats. Parameters for the three routes of administration were estimated using non-compartmental analysis using WinNonlin (version 1.5, Pharsight Inc., CA). C max is the maximum observed drug concentration and AUC 0-360min is the area under the curve in a given tissue. Data are expressed as mean ± SD for n = 4. * indicates p<0.05 compared to other two groups.

Discussion

This is the first study to demonstrate suprachoroidal injection in a rat model and compare the pharmacokinetics of suprachoroidal injection with intravitreal and posterior subconjunctival injections using noninvasive ocular fluorophotometry. We demonstrated that 1) sodium fluorescein levels can be monitored noninvasively in different ocular tissues after suprachoroidal, posterior subconjunctival, and intravitreal injections in rats using ocular fluorophotometry; 2) the suprachoroidal route is the most effective method for attaining high concentrations of sodium fluorescein in the choroid-retina region; and

3) the rate and extent of delivery to the choroid-retina is highest with suprachoroidal injection.

Possible reasons for autofluorescence and broad vs. sharp NaF peaks in different regions

Baseline Fluorotron scans showed very minimal autofluorescence peaks in the choroid-retina, lens, and cornea regions (Figure 5.2A). A very low autofluorescence was also observed in the anterior chamber. Possible reasons for autofluorescence from these tissues are the presence of fluorescent nucleotides and lipid metabolites (Eldred, Miller et al. 1982; Fariss, Apte et al. 1997; Van Schaik, Alkemade et al. 1999). Autofluoresence in the choroid-retina region of rats is attributed to the presence of lipofuscin granules

(Eldred, Miller et al. 1982; Katz, Robison et al. 1984) in the retinal pigment epithelial cells and elastin layer in the bruch’s membrane (Fariss, Apte et al. 1997).

Autofluoresence in the lens can be due to the presence of flavoproteins such as FMN in the lens epithelium (Tsubota, Laing et al. 1987). Rat corneal autofluorescence is caused by pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH)

(Lee, Schade et al. 1985) and flavin nucleotides such as flavin mononucleotide (FMN)

(Batey, Daneshgar et al. 1992) in metabolically active cells such as the corneal epithelium and endothelium (Van Schaik, Alkemade et al. 1999). Baseline autofluorescence and peak assignments are shown in Figure 5.2A.

Using fluorophotometry, we compared NaF levels in the eye after suprachoroidal, subconjunctival, and intravitreal injections. The signals observed were much higher than the background fluorescence and each route resulted in peak signals at a distinct location, corresponding to the site of injection. Suprachoroidal injection of NaF in the rat eye showed a broad peak (Figure 5.2B) possibly due to the ‘halation’ of the choroid-retina response (Gray, Mosier et al. 1985). Halation or secondary fluorescence occurs due to the presence of a highly autofluorescent tissue such as choroid near the point of quantification. Light passing straight through the choroid- retina is reflected back by the choroid base and scattered around. This causes the fluorescence to bleed through and results in tailing of the choroid-retina response. Similar to suprachoroidal injection, the peak attained after subconjunctival injections was also broad (Figure 5.2C). In the case of intravitreal injections, a comparatively sharper peak was attained (Figure 5.2D), primarily due to the lack of any secondary fluorescence.

Higher rate and extent of delivery to choroid-retina after suprachoroidal injection

NaF levels in the anterior chamber: We compared the pharmacokinetics of NaF after suprachoroidal, subconjunctival, and intravitreal injections (Figure 5.3-5.6). In pharmacokinetic analysis, the rate and extent of delivery is related to C max and AUC.

Further, T max is related to the rate of delivery, if the elimination remains the same in a

given tissue, irrespective of the route of drug entry and concentration. Rapid absorption into the choroid-retina region, as indicated by the T max value, was observed immediately upon suprachoroidal injection. In comparison, intravitreal and subconjunctival injections had C max at 27.5 and 10 minutes, respectively. After suprachoroidal injection, the C max of

NaF in choroid-retina was 36- and 25- fold higher than subconjunctival and intravitreal injection, respectively. Further, the extent of delivery was also 6- and 2- fold higher than subconjunctival and intravitreal injections, respectively. The higher rate and extent of delivery to choroid-retina after suprachoroidal injection is due to targeted deposition of the dose in the choroid. In comparison to suprachoroidal injection, drug molecules administered by intravitreal and subconjunctival routes need to cross various barriers to reach the target tissue. Olsen et al. (Olsen, Feng et al. 2011) have also shown localized delivery to choroid-retina following suprachoroidal injection of bevacizumab.

Araie and Maurice (Araie and Maurice 1991) froze and sectioned eyes to expose the vitreous and fixed them to a cryotome stage. The entire vitreous was scanned using a fluorometer. Readings obtained by the fluorometer were normalized by assigning a maximum value behind the lens at 100. Lines were drawn for the values 90, 80, and so on, obtained by interpolation between the measured values. A concentration contour map was created based on the measured values. Araie and Maurice observed that NaF concentration was the highest in the vitreous immediately adjacent to the lens and dropped to a low value of 30 over the entire surface of the retina and iris-ciliary body indicating that the majority of NaF was cleared from the anterior segment (Araie and

Maurice 1991). Thus, following intravitreal injections, NaF is expected to predominantly clear through the anterior segment (Maurice 1976). Therefore, NaF detected in the

choroid-retina region after intravitreal injection in our study can be due to accumulation of NaF at the retinal surface (Araie and Maurice 1991) and may not truly depict delivery to the choroid-retina region.

Posterior subconjunctival injection in rats, analogous to sub-Tenon injection in humans, is expected to deliver drugs to the back of the eye tissues(Raghava, Hammond et al. 2004). The extent of delivery to choroid-retina after subconjunctival injection is lower compared to suprachoroidal route in our study, possibly due to multiple clearance pathways. In comparison to subconjunctival injection, suprachoroidal injection places the entire dose in close proximity to the choroid, thereby resulting in higher drug levels and exposure to the choroid-retina region. Following subconjunctival injection, the drug may encounter several elimination pathways including episcleral and conjunctival vasculature prior to entering the choroid (Robinson, Lee et al. 2006).

NaF exposure to the vitreous: Both C max and AUC for vitreous humor delivery of NaF were in the order: intravitreal injection > suprachoroidal > subconjunctival. This rank order is consistent with the proximity of vitreous to the site of administration. The further removed the dose was from the vitreous, the lower the drug delivery.

NaF exposure to anterior chamber: In our study, we detected very low levels of

NaF in the anterior chamber region after suprachoroidal, subconjunctival, or intravitreal injection when compared to NaF levels in other tissues. Following suprachoroidal injection, anterior chamber C max was significantly higher than intravitreal injection and subconjunctival injection, with the rank order being: suprachoroidal > intravitreal > subconjunctival. A similar rank order was observed for NaF exposure in the anterior chamber. Contrary to our observations, following suprachoroidal injections in ex vivo

porcine eyes, Seiler et al. (Seiler, Salmon et al. 2011) could not detect any signal for contrast agent in the anterior segment region. This may be because following suprachoroidal and subconjunctival injections (Robinson, Lee et al. 2006), clearance occurs immediately due to the proximity of blood vessels when compared to intravitreal injections. Therefore, very low quantities of NaF reach the anterior chamber after suprachoroidal and subconjunctival injections. The suprachoroidal injections in our study may be more anterior compared to earlier studies, resulting in significant NaF exposure to the anterior segment. Additionally, the sensitivity of detection of contrast agents may not have been sufficient in the earlier study (Seiler, Salmon et al. 2011) to pick up the signal from the anterior segment following suprachoroidal injection. Future studies need to assess the influence of site of suprachoroidal injection on drug distribution.

NaF clearance by various routes: Although the half-lives for the terminal declining phase of concentration-time profiles could not be estimated for various tissues due to fluctuations in the signal in the terminal regions, the time for NaF levels to approach baseline values in choroid-retina was in the following rank order: intravitreal > suprachoroidal > subconjunctival. While the rapid approach to baseline with subconjunctival route can be attributed to lowest drug delivery by this route, slow approach to baseline with intravitreal route is most likely due to slow absorption of the drug to the choroid from the vitreous humor. NaF in choroid is expected to be eliminated by the same pathways irrespective of the mechanism of drug entry/administration. Also, the elimination kinetics are expected to be the same irrespective of the route of administration, unless the elimination pathways are affected by drug concentrations.

Once in the choroid, NaF be removed rapidly due to choroidal blood flow. Rapid drug

clearance from choroid is empirically attributed to high blood flow. The blood flow velocity in the human choroid (1-1.2 ml/min ; 0.052-0.198 m/s (Polska, Polak et al.

2004)) is several fold lower than the blood flow (1175-2110 ml/min (Bradley, Ingelfinger et al. 1945); 0.585-0.766 m/s (Hubner, Steudel et al. 2000)) in the liver, a primary organ for drug clearance. However, after tissue weight normalization the choroidal blood flow is significantly higher than the hepatic blood flow. Previous studies indicated that tissue weight normalized blood flow to the human choroid and liver were 1200 ml/100 gm tissue/min (Friedman, Kopald et al. 1964) and 1.7 ml/100gm/min (Carlisle, Halliwell et al. 1992), respectively. Thus, although the total blood flow per unit time and the velocity of the blood in choroid are much lower compared to the liver, the blood supply per unit tissue weight is much higher in the choroid than the liver. However, it is unclear how these differences in blood flow play a role in choroid clearance of solutes. For liver clearance of drugs, total blood flow is taken into consideration (Riedel 2006). Given the much lower total blood flow in the choroid, it is anticipated that the clearance in choroid would be much less compared to the liver, especially for drugs with high extraction ratio.

However, one of the limitations of ocular fluorophotometry is that this technique cannot be used for drug molecules that are not fluorescent similar to fluorescein. Therefore, most drug molecules require a fluorescein-like tag to be monitored by fluorophotometry.

However, such tags may alter physicochemical properties of small solutes and drugs, thereby potentially altering their rate and/or extent of delivery to the eye tissues.

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Conclusions

In summary, this study shows that the suprachoroidal injection is the most effective route for localized delivery of therapeutics to the choroid-retina region. Further, in this study we have also demonstrated the applicability of ocular fluorophotometry for non-invasive monitoring of drug levels following administration by various routes.

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CHAPTER VI

LIGHT ACTIVATED, IN SITU FORMING GEL FOR SUSTAINED

SUPRACHOROIDAL DELIVERY OF BEVACIZUMAB 2

Abstract

Purpose

To develop a light activated polycaprolactone dimethacrylate and hydroxyethyl methacrylate based gel network that sustains the release of stable, active bevacizumab (an anti-VEGF antibody used to treat choroidal neovascularization) and to assess sustained ex vivo delivery in rabbit eyes and in vivo delivery in rat eyes following in situ gel formation in the suprachoroidal space.

Methods

Polycaprolactone dimethacrylate (PCM) was synthesized from polycaprolactone diol (PCD) and evaluated using NMR spectroscopy. PCM was used to cross-link hydroxyethyl methacrylate (HEMA) in the presence of 365 nm UV light and 2, 2- dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator. Bevacizumab was entrapped in the gel using 3 different cross-linking durations of 3, 7, and 10 minutes. In vitro release of bevacizumab in PBS pH 7.4 at 37ºC during a 4 months study was quantified using a VEGF-binding based ELISA. Stability of released bevacizumab was monitored by size exclusion chromatography (SEC) and circular dichroism (O'Brart,

2 Puneet Tyagi, Matthew Barros, Jeffrey W. Stansbury, and Uday B. Kompella (2013) Light-Activated, In Situ Forming Gel for Sustained Suprachoroidal Delivery of Bevacizumab, Mol. Pharmaceutics, 2013, 10 (8), pp 2858–2867 102

Kwong et al.). Alexa Fluor ® 488 dye conjugated bevacizumab mixed with polymers was injected suprachoroidally in rabbit eyes to study the effect of different cross-linking durations on the spread of the dye conjugated bevacizumab. In vivo delivery was assessed in Sprague Dawley (SD) rats by injecting Alexa Fluor ® 488 dye conjugated bevacizumab mixed with polymers followed by cross-linking for 10 minutes. Spread in the rabbit eyes and in vivo delivery in rat eyes was monitored noninvasively using a fundus camera and Fluorotron Master™.

Results

Formation of PCM was confirmed by the disappearance of hydroxyl peak in

NMR spectra. Cross-linking duration of 10 minutes resulted in a burst release of 21 % of bevacizumab. Other cross-linking durations had ≥ 62 % burst release. Bevacizumab release from 10 minute cross-linked gel was sustained for ~ 4 months. Release samples contained ≥ 96.1 % of bevacizumab in the monomeric form as observed in SEC chromatograms. Circular dichroism confirmed that secondary β-sheet structure of bevacizumab was maintained after release from the gel. As the cross-linking duration was increased to 10 minutes, the gel/antibody was better confined at the injection site in excised rabbit eye suprachoroidal space. Delivery of Alexa Fluor ® 488 dye conjugated bevacizumab was sustained for at least 60 days in the suprachoroidal space of SD rats.

Conclusion

PCM and HEMA gel sustained bevacizumab release for 4 months and maintained the stability and VEGF-binding activity of bevacizumab. Light activated PCM and

HEMA gel is suitable for in situ gel formation and sustained protein delivery in the suprachoroidal space.

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Introduction

Intravitreal injections of bevacizumab, an anti-VEGF full-length antibody, are being used off-label for the treatment of choroidal neovascularization. Bevacizumab as well as the FDA approved intravitreal therapy with ranibizumab, a Fab fragment of bevacizumab, require monthly injections due to the chronic nature of the disease.

However, repeated intravitreal injections can elevate the risk of infection, retinal detachment (Arevalo, Maia et al. 2008), and hemorrhage (Fung, Rosenfeld et al. 2006).

Thus, there is a need to develop a slow release system for therapeutic proteins such as bevacizumab, in order to maintain therapeutically relevant concentrations over extended periods. Numerous sustained delivery approaches including liposomes (Kim, Choi et al.

1998), polymeric micro- and nano- particulate systems (Anderson, Ozaki et al. 2001;

Genta, Perugini et al. 2001; Sinha and Trehan 2003), mesoporous silica films(Andrew,

Anglin et al. 2011), and gels (Aimetti, Machen et al. 2009; Guziewicz, Best et al. 2011;

Koutsopoulos and Zhang 2012) have been assessed to sustain protein drug delivery.

However, to our knowledge, there are no reports demonstrating sustained release of an antibody drug in its stable form for 4 months. This is because most techniques for preparing polymeric delivery systems adversely affect protein stability.

Formulation of sustained delivery systems such as microparticles and nanoparticles introduce organic solvent-water and air-water interfaces, that are major sources of protein denaturation and aggregation (Alonso, Gupta et al. 1994; Lu and Park

1995; Cleland and Jones 1996; Kim and Park 1999; Sah 1999; Sah 1999). Furthermore, sonication used during particle preparation can reduce protein stability (Suslick 1986;

Bittner, Morlock et al. 1998; Zambaux, Bonneaux et al. 1999). Liposomes, especially

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small vesicles employ sonication, which can be detrimental for protein stability (Lentz,

Madden et al. 1982). In comparison to particulate systems and liposomes, gel systems are more attractive and offer a promising delivery platform due to their simplicity in formulation, and solvent free condition. Polymeric gels are useful in a wide range of applications such as drug delivery (Lu and Anseth 1999; Cai, Liu et al. 2005), dentistry

(Stansbury and Dickens 2001), and tissue engineering (Lee and Robinson 2001). In situ forming gels based on a stimuli-response are particularly attractive for ophthalmic application (Christie and Kompella 2008). To form such gels in situ, a polymeric mixture can potentially be injected through a small gauge needle in a confined space within the eye, followed by activation using a stimulus such as light. This is beneficial since viscous gels might require large bore needles and relatively prolonged duration of injection.

We aimed to develop a light activated gel using a polycaprolactone-derivatized dimethacrylate (PCM) and hydroxyethyl methacrylate (HEMA) for sustaining the release of bevacizumab (Avastin™, Genetech Inc, CA), a 145 kDa monoclonal antibody, in its active form. PCM has been used extensively in drug delivery systems (Goodwin, Braden et al. 1998; Aishwarya, Mahalakshmi et al. 2008; Waknis and Jonnalagadda 2011), primarily due to its slow degradation rate (Kweon, Yoo et al. 2003; Lam, Savalani et al.

2008). Other advantages of using PCM are that it does not create an acidic environment

(a drawback of solid poly (lactic acid) and poly (glycolic acid) microspheres for protein drugs), is biocompatible, and can easily be blended with a wide variety of polymers.

Similar to PCM, HEMA also has a number of biomedical applications, including manufacturing of soft contact lenses (Alvarez-Lorenzo, Hiratani et al. 2002), dental adhesives (Moraes, Garcia et al. 2012), and drug delivery systems (Dziubla, Torjman et

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al. 2001; Hsiue, Guu et al. 2001), due to its hydrophilicity and high water uptake capacity. Furthermore, both polycaprolactone and HEMA are present in FDA approved products for human use.

Bevacizumab was chosen in our study because of its similar efficacy to ranibizumab, an FDA approved drug product, in the treatment of choroidal neovascularization (CNV) associated with wet age-related macular degeneration (AMD)

(Fine, Zhitomirsky et al. 2009). Wet AMD, the leading cause of blindness in the USA, is prevalent in 8 million people and is a lifelong condition. However, bevacizumab, which targets angiogenesis pathways by binding with vascular endothelial growth factor

(VEGF), has a short vitreous half-life of 4.8 days (Bakri, Snyder et al. 2007), requiring repeated injections. Therefore, a sustained delivery system for bevacizumab would be of immense clinical significance.

In addition to the development of a sustained delivery gel system for bevacizumab, we also optimized its administration via suprachoroidal route in excised rabbit eyes and confirmed in vivo sustained delivery of the gel system in the suprachoroidal space of SD rats (Tyagi, Kadam et al. 2012). Suprachoroidal delivery is a novel route that is less invasive to the retina, than intravitreal injections. Suprachoroidal injections localize the therapeutic agents adjacent to the choroid region, the target tissue affected in CNV (Patel, Lin et al. 2011). However, a drawback in suprachoroidal delivery is that due to abundance of blood vessels in the choroid region, bevacizumab is cleared very rapidly following suprachoroidal administration (Olsen, Feng et al. 2011). Thus, the gel systems developed in this study are of potential value in sustaining bevacizumab levels in the choroid.

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Materials and methods

Materials

Polycaprolactone diol (Mn= 530), 2-isocyanatoethyl methacrylate, hydroxyethyl methacrylate (HEMA), dibutyltin dilaurate, 2,2dimethoxy-2-phenylacetophenone

(DMPA), and bovine serum albumin were purchased from Sigma Aldrich, St Louis, MO.

Alexa Fluor ® 488 protein labeling kit was purchased from Invitrogen Inc., Grand Island,

NY. ELISA plates were purchased from Corning Incorporated, Tewksbury, MA.

VEGF165 and secondary antibody for ELISA was purchased from R&D Systems, Inc,

Minneapolis, MN.

Preparation and characterization of polycaprolactone dimethacrylate (PCM)

Polycaprolactone diol was reacted with 2-isocyantoethyl methacrylate at 1:2 molar ratios in the presence of a trace amount of dibutyltin dilaurate as a catalyst to create polycaprolactone dimethacrylate. The product was characterized by 1H NMR using a

Varian Inova 500 MHz NMR (Agilent Technologies, Santa Clara, CA).

Preparation of PCM-HEMA gel

A co-monomer mixture was prepared with a ratio of 90:10 (45 µl and 5 µl of

HEMA: PCM). Bevacizumab (50 µl of a 25 mg/ml solution) was added to the above solution. Photoinitiator (DMPA) was added to the dispersion and vortexed for 1 minute.

The copolymerization was initiated by irradiation with near-UV light at 365 nm using

Acticure® spot curing system (EXFO Electro-Optical Engineering Inc, Richardson, TX), positioned 8 cm from the dispersion. The intensity of the UV light was 0.03 W. Gels were prepared at three different durations of light exposure to yield different degrees of cross-linking (3, 7, and 10 minutes).

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To assess the degree of cross-linking, conversion data were obtained using a

Nicolet 6700 FT-IR spectrometer (Thermo Scientific, IL) operating in the near-infrared region. The liquid monomer sample was placed between two glass cover slips and placed in a block, with an entry and exit port for the infrared beam, and photopolymerized at room temperature. The thickness of the liquid monomer sample between the cover slips was 0.8 mm and the diameter of the sample exposed to infrared beam was 1 cm. The intensity of the band at around 6150 cm -1 (Hill, Perera et al. 1997), arising from the reacting =CH 2 double bond, was used to quantify the degree of conversion, which correlates with the cross-link density in the gel.

In vitro sustained release of bevacizumab from gel

Gels prepared at different cross-linking durations were studied for release of bevacizumab at 37 °C. The method used to follow drug release from the gel was adopted from Zhang et al.(Zhang, Parsons et al. 2002). Briefly, 1 ml of PBS buffer pH 7.4 was carefully layered over the surface of the gel matrix. The gel matrix volume was 100 µl and the amount of bevacizumab entrapped in the gel matrix was 1.25 mg. At predetermined time intervals the entire 1 ml was removed from the surface of the gel and quantified for bevacizumab released. Fresh 1 ml PBS pH 7.4 was layered over the gel for continuation of the release study. Samples were evaluated for the amount of bevacizumab by using ELISA as described below. Release was calculated based on the initial amount of bevacizumab entrapped in the gel (1.25 mg). The comparisons of the mean between the three groups were performed using one-way ANOVA followed by Tukey's post hoc analysis (GraphPad Prism, GraphPad Software, Inc., CA). The differences were considered statistically significant at P < 0.05.

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Quantification of bevacizumab released from gel

ELISA was used to quantify bevacizumab in the release samples. All release samples were diluted in PBS pH 7.4 buffer. A 96-well plate was coated with 100 µl per well of 0.2 µg/ml of VEGF165 and incubated for 14 h. The plates were later blocked by adding 300 µl per well of reagent diluent (prepared by dissolving 1% bovine serum albumin in PBS pH 7.4). Bevacizumab standard or samples were added to each well and incubated for 2 hours at room temperature. The addition of 100 µl of secondary antibody

(diluted 1:5000 in 0.1 % bovine serum albumin) to each well was followed up by incubation for 2 h at room temperature. TMB substrate solution (100 µl) was added to each well and plates incubated in the dark for 30 minutes at room temperature. Lastly, 50

µl of stop solution (0.5 M HCl) was added to each well and plate gently tapped to ensure thorough mixing. The optical density of each well was immediately determined, using a microplate reader (Molecular Devices Corp., CA) set to 450 nm. Avastin formulation was used for preparing the standard curve. Three washings were performed at each step, after addition of release sample, blocking buffer, VEGF standard, detection antibody, and streptavidin-HRP. The wash buffer was made by dissolving 0.05% Tween 20 in PBS pH

7.4 buffer.

Stability characterization by size exclusion chromatography (SEC)

A size exclusion column (TSK ® Gel G3000SWX) was attached to high performance liquid chromatography (Waters Corporation, Milford, MA). A UV detector scanning over the wavelength of 210-400 nm was used to detect the output from the size exclusion column. The mobile phase was an aqueous solution of 0.182 M KH 2PO 4, 0.018

M K 2HPO 4 and 0.25 M KCl at pH 6.2. Flow rate of the mobile phase was 0.50 ml

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/minute. A standard of bevacizumab was prepared by dilution from a stock solution of 25 mg/ml to 50 µg/ml, which is similar in concentration to the marketed formulation of bevacizumab. The solution was prepared using 60 mg/mL trehalose, 5.8 mg/mL

NaH 2PO 4, 1.2 mg/mL Na 2HPO 4 and 0.4 mg/mL polysorbate 20 at pH 6.2. The volume of the injection was 100 µl. All samples were centrifuged at 14,000 g for 10 minutes before use.

Secondary structure characterization by circular dichroism

Bevacizumab released from the cross-linked gel was studied in the "far-UV" spectral region (190-250 nm) by circular dichroism to evaluate the loss of the secondary structure of bevacizumab after entrapment and release from the gel. The spectra were obtained on an AVIV model 62 DS spectropolarimeter (AVIV Biomedical, Inc.,

Lakewood Township, NJ). Protein solutions were transferred into a 1 mm path length quartz cell, which was placed in a thermostatic cell holder. Data were collected at 1 nm intervals utilizing a 2 nm bandwidth. The spectrum for the appropriate formulation blank was collected and subtracted from each protein formulation spectrum.

Suprachoroidal injection in ex vivo rabbit eyes

To assess the retention of the polymer at different cross-linking durations, bevacizumab was conjugated to Alexa Fluor ® 488 dye using Alexa Fluor 488 ® protein labeling kit. The procedure for conjugation was followed as per the vendor’s protocol. A solution was prepared using PCM and HEMA monomers in a ratio of 90:10 (45µl and

5µl of PCM: HEMA). Alexa Fluor ® 488 dye conjugated bevacizumab (50 µl) was added to the above solution. Photoinitiator was added to the dispersion and vortexed for 1 minute. The above dispersion (5 µl) was injected in the suprachoroidal space of ex vivo

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rabbit eyes (n=3 for each group) using a 50 µl Hamilton glass syringe attached to a 30G

5/4” needle. Eyes were subjected to near-UV light at 365 nm at three different cross- linking durations (3, 7, and 10 minutes) at 0.03W intensity. Fluorescence was studied up to 48 h after injection using Fluorotron Master™ (Ocumetrics, Inc, Mountain View, CA) and fundus camera (Genesis Df, Kowa Optimed Inc, Torrance, CA).

In vivo sustained delivery in SD rats

All animals were treated according to the Association for Research in Vision and

Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision

Research. Animal protocols followed during this study were approved by the Institutional

Animal Care and Use Committee of the University of Colorado Anschutz Medical

Campus, Aurora, CO. Adult male Sprague Dawley rats (150–180 gm) were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN, USA). Rats were anesthetized using an intraperitoneal injection of a mixture of 80 mg/kg ketamine and 10 mg/kg xylazine.

A solution was prepared using PCM and HEMA monomers in a ratio of 90:10 (45 µl and

5 µl of PCM: HEMA). Alexa Fluor ® 488 dye conjugated bevacizumab (50 µl of a

1mg/ml solution) was added to the above solution. Photoinitiator was added to the dispersion and vortexed for 1 minute. Alexa Fluor ® 488 dye conjugated bevacizumab along with the gel forming polymers (5 µl) was injected in the suprachoroidal space of

SD rats (n=4) using a 10 µl Hamilton glass syringe attached to a 34G 1/2” needle. Eyes were subjected to near-UV light at 365 nm for 10 minutes. We used 10 minute exposure at an exposure intensity of 3.18 mW/cm2 in our in vivo study. UV light at 365 nm exposure for 30 minutes at an exposure intensity of 3.0 ± 0.3 mW/cm2 has been used in human clinical trials to treat keratoconus and bacterial keratitis (Makdoumi, Mortensen et

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al. 2012; Viswanathan and Males 2012; O'Brart, Kwong et al. 2013). Fluorescence was studied after injection using Fluorotron Master™ (Ocumetrics, Inc, Mountain View, CA) and fundus camera (Genesis Df, Kowa Optimed Inc, Torrance, CA).

In vitro toxicity

ARPE (human retinal pigment epithelial) cells (passage#24) were plated in a 96- well plate at a seeding density of 10000 cells/well and allowed to adhere to the well for

24 hours. After 24 hours, cells were incubated with the media only (control), gel forming polymers (PCM, HEMA, and photoinitator) without cross-linking, gel forming polymers cross-linked for 3 minutes, 7 minutes, or 10 minutes. At the end of 24 hours, the media was aspirated out and 200 µl fresh serum free medium was added to each well. MTT reagent (Sigma Aldrich, MO) i.e. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) , (20 l of 5 mg/ml MTT dissolved in PBS pH 7.4) was added to each well and incubated at 37°C for 3 hours. The medium was aspirated out and the formazan crystals formed were dissolved in 200 l of dimethyl sulfoxide. The absorbance of the color developed was measured at 570 nm using a microplate reader.

In vivo toxicity

A solution was prepared using PCM and HEMA monomers in a ratio of 90:10 (45

µl and 5 µl of PCM: HEMA). Alexa Fluor ® 488 dye conjugated bevacizumab (50 µl of a

SD rats (n=4 eyes) using a 10 µl Hamilton glass syringe attached to a 34G 1/2” needle.

Eyes were subjected to near-UV light at 365 nm for 10 minutes. At the end of 40 days of

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injection, rats were euthanized and eyes enucleated. Eyes were fixed in Davidson’s fixative for 24 hours and 5 µm thick sections were attained. The sections were stained using hematoxylin and eosin and examined under a light microscope (Olympus BX41 laboratory microscope) fitted with a camera (Diagnostics Instruments, Inc.).

Results

Preparation and characterization of polycaprolactone dimethacrylate (PCM)

Figure 6.1 shows the NMR spectra for polycaprolactone diol, isocyanatoethyl methacrylate, and polycaprolactone dimethacrylate (Figure 6.1A, 6.1B, and 6.1C, respectively). As the reaction between polycaprolactone diol and isocyanatoethyl methacrylate completed, the -OH group peaks, seen at 3.64 and 3.70 ppm, (Figure 6.1A) disappeared and the -NH peaks at 5.01 and 5.15 ppm appeared (Figure 6.7C). The methacrylate peaks from isocyanatoethyl methacrylate were also seen in the final spectrum at 5.60 and 6.12 ppm (Figure 6.1C).

Preparation of gel

Figure 6.2 shows the reaction scheme for the cross-linking of PCM with HEMA.

In the presence of 365 nm UV light, DMPA photoinitiator was converted to benzoyl free radical, which further attacked the =CH 2 group in PCM and HEMA. This resulted in propagation via chain addition, which consumed the free monomer. FT-IR spectra confirmed the disappearance of ~ 78 % of the =CH 2 peak area (wavenumber of ~ 6150 cm -1) at the end of 10 minutes.

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Figure 6.1: NMR spectra for (A) polycaprolactone diol, (B) isocyanoethyl methacrylate, and (C) polycaprolactone dimethacrylate. Formation of polycaprolactone dimethacrylate was confirmed by the appearance of the NH peaks at 5.01 and 5.15 ppm. Methacrylate peaks were evident at 5.60 and 6.12 ppm.

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Figure 6.2: Reaction scheme for the preparation of cross-linked gel. Polycaprolactone dimethacrylate was cross-linked to hydroxylethyl methacrylate in the presence of 365 nm UV light and photoinitiator (2,2-dimethoxy-2-phenyl-acetophenone).

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In vitro sustained release of bevacizumab from cross-linked gel

Figure 6.3 shows the release of bevacizumab from the gel at different cross- linking durations. When reacted for either 3 or 7 minutes, the resulting gels exhibited a burst release of 82 % (± 6.8 %) and 62 % (± 3.5 %) of the drug, respectively. With the increase in the cross-linking duration to 10 minutes, the initial burst release was decreased to 21 % (± 0.4 %). With 10 minute cross-linking, bevacizumab activity/delivery in the release medium was sustained up to 4 months. Release from gel cross-linked for 3 minutes was significantly different from release from gel cross-linked for 10 minutes at 0.25 hours. Release from gel cross-linked at 3, 7, and 10 minutes was significantly different from each other from 0.50 hours to 30 days. The release data for gel cross-linked for 10 minutes was significantly different from the other two gels from

40 to 90 days. However, there was no significant difference between release from gels cross-linked at 3 and 7 minutes from 40 to 90 days. The release from gels cross-linked at

3, 7, and 10 minutes was not significantly different from each other beyond 90 days.

Physical stability of bevacizumab released from cross-linked gel

Figure 6.4 shows size exclusion spectra of bevacizumab after release from the gel.

The chromatogram of standard bevacizumab formulation showed a primary peak at retention time of ~ 8.5 minutes. A peak at ~ 7.2 minutes indicated dimers or trimers and accounted for 1.5 % of the total peak area in control sample. Peaks at ~ 8.5 and ~ 7.2 minutes were also observed in release samples at all time points (1, 2, 3, and 4 months).

However, fragments of bevacizumab were observed in the release samples. A peak at ~

10.2 minutes increased from 0.8 % in 1 month release sample to1.8 % in the 4 month release samples.

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Figure 6.3: Cumulative release of bevacizumab from the gel at three different cross- linking durations. Burst release from the gel prepared by 10 min cross-linking was 21%. Other cross-linking durations had ≥62% burst release. Data are expressed as mean ± SD for n = 3.

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Figure 6.4: Size exclusion chromatograms of bevacizumab released from the gel prepared by 10 min cross-linking. Marketed formulation of bevacizumab (Avastin, Genentech Inc., CA) was diluted to 50 g/mL and used as a control.

Conformational stability of bevacizumab released from cross-linked gel

Figure 6.5 gives the ellipticity spectra of bevacizumab released from gel. The chromatogram was collected in the far UV-spectral range (190-250 nm). A negative band between 210 and 220 nm is a signature of β-sheet in bevacizumab. The sample released from the cross-linked gel also showed the characteristic band, indicating that bevacizumab did not lose its secondary β-sheet structure after entrapment in the cross- linked gel.

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Figure 6.5: Circular dichroism spectra for of bevacizumab released from the gel prepared by 10 min cross-linking. The marketed formulation of bevacizumab (Avastin, Genentech Inc.,CA) was diluted to 50 g/mL and used as a control.

Retention of Alexa Fluor ® 488 dye conjugated bevacizumab in ex vivo eyes

Alexa Fluor ® 488 dye conjugated bevacizumab when entrapped in PCM and

HEMA cross-linked for 10 minutes, maintained significantly higher levels at the site of injection (up to 48 hours) as compared to 3 and 7 minutes cross-linking. Peak levels after

10 minutes cross-linking reduced by 36% in 60 minutes and then the levels were sustained till 48 hours. Fluorescence levels returned to the level of blank within 8 and 16 h for 3 and 7 minutes cross-linking durations, respectively. Fundus images (Figure 6.6A) depicted that cross-linked gel maintained its shape even at the end of 48 hours in the eyes with 10 minutes cross-linking. In comparison, fundus images of eyes with 3 and 7 minutes cross-linking did not show any fluorescence beyond 8 and 16 hours, respectively.

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Figure 6.6: Injection and in situ cross-linking of gel-forming polymers mixed with Alexa Fluor 488 dye conjugated bevacizumab, which is feasible after injection in suprachoroidal space. (A) Fundus camera images depicting the effect of cross-linking duration on spread of Alexa Fluor 488 dye conjugated bevacizumab in excised rabbit eyes. (B) Ten minute cross-linking duration resulted in sustained levels of Alexa Fluor 488 dye conjugated bevacizumab for more than 48 h, as assessed by ocular fluorophotometry. In comparison, 3 and 7 min cross-linking duration could only confine Alexa Fluor 488 dye conjugated bevacizumab for 8 and 16 h, respectively. A portion of 5 L of the gel-forming polymers containing Alexa Fluor 488 conjugated bevacizumab was injected in the suprachoroidal space of ex vivo rabbit eyes using a 50 L Hamilton glass syringe attached to a 30G needle. Eyes were subjected to near UV light at 365 nm at 3, 7, and 10 min cross-linking durations. Data are represented as mean ± SD for n = 3.

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In vivo sustained delivery in SD rats

Delivery of Alexa Fluor ® 488 dye conjugated bevacizumab was sustained for 60 days after entrapment in gel network. Figure 6.7A shows representative fundus camera images of SD rat eyes that were injected with Alexa Fluor ® 488 dye conjugated bevacizumab and gel forming polymers which were cross-linked in situ using 365 nm

UV light. In comparison to eyes wherein the gel forming polymer was cross-linked, eyes without cross-linked gel lost the fundus camera signal by the end of day 5. Fluorescence of Alexa Fluor ® 488 conjugated bevacizumab decreased by ~ 34 % from a concentration of 50.3 µg/ml (± 15.7) immediately after injection to 33.4 µg/ml (± 16.4) at the end of day 1 following entrapment in cross-linked gel. In comparison, ~ 85 % of the fluorescence was lost in the eyes wherein the gel forming polymers were not cross-linked and the fluorescence reached baseline levels by the end of day 5.

In vitro toxicity (Figure 6.8)

In the absence of cross-linking, mixture of gel forming polymers and photointiator were found to induce 27.6% cell death in ARPE cells when compared to control.

Following cross-linked gel exposure, it was observed that the cell death reduced to 23.8,

20.3, and 5.3% following incubation with gel cross-lined for 3, 7, and 10 minutes, respectively. This indicates that the toxicity is primarily caused by uncross-linked polymers and free photoinitiator. However, as the individual polymers and photoinitiator is used up, toxicity is reduced.

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A Immediate 1 day 5 days 10 days

Control

20 days 30 days 40 days 60 days

8 Control

c .o-~ 70 rcs E 60 + Cross-linked gel EC, + Gel without cross-linking ~ ::::1. so N~ - -Blank eye ·-(.) ~ 40 C'O s::: ~ G) 30 .Q(.) • en ~! 20 Q) 0 - ~ 10 C:S:- '1- 0 0 20 40 60 80

Time (days}

Figure 6.7: Alexa Fluor 488 dye conjugated bevacizumab retained in the suprachoroidal space of rats up to 60 days following entrapment in a gel.

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Figure 6.7: Alexa Fluor 488 dye conjugated bevacizumab retained in the suprachoroidal space of rats up to 60 days following entrapment in a gel. (A) Fundus camera images of SD rat eyes at different time points after injection of Alexa Fluor 488 dye conjugated bevacizumab and gel-forming polymers in the suprachoroidal space. Gels were further cross-linked in situ using 365 nm UV light. (B) In comparison, Alexa Fluor 488 dye conjugated bevacizumab levels decreased rapidly, and no signal was attained beyond 5 days, wherein gel-forming polymers were not cross-linked. (C) Ocular fluorophotometry (Fluorotron Master) was used to determine the levels of Alexa Fluor 488 dye conjugated bevacizumab following injection of Alexa Fluor 488 dye conjugated bevacizumab and gel-forming polymers in the suprachoroidal space and cross-linking. After an initial decrease, constant levels of Alexa Fluor 488 dye conjugated bevacizumab were maintained in the suprachoroidal space where gel-forming polymers were cross-linked. Data are expressed as mean ± SD for n = 4. Blank eye levels (dashed line) were measured once before the study and extrapolated to remaining time points.

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Figure 6.8: Cross-linked gel for 10 min, which was not toxic in vitro. In vitro toxicity was assessed by exposing human retinal pigment epithelial (ARPE) cells to the gel- forming polymers either without cross-linking or after cross-linking for 3, 7, or 10 min. Key: PCM—polycaprolactone dimethacrylate, HEMA—hydroxyethyl methacrylate, DMPA—2,2-dimethoxy-2-phenylacetophenone. PCM+HEMA+DMPA is a mixture of the gel-forming polymers without cross-linking (2nd bar). The time in brackets indicates the time for cross-linking in the three groups. For control, only media was used and considered as 100% viability. Data are represented as mean ± SD for n = 4.

In vivo toxicity (Figure 6.9)

According to histological examination of the anterior section of the SD rat eye at the end of 40 days of injection of gel forming polymers and Alexa Fluor ® 488 conjugated bevacizumab followed by in situ cross-linking , no morphological or structural changes were observed. This confirmed that the gel cross-linked for 10 minutes did not induce any gross toxicity in the ocular tissues.

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Figure 6.9: Gel cross-linked for 10 min, which did not induce any retinal detachment, atrophy, or hypertrophy. In vivo toxicity was assessed following (A) suprachoroidal injection of the gel-forming polymers in SD rats and further cross-linking in situ using 365 nm UV light. (B) Rat eyes without any injection were used as control. At the end of 40 days rats were euthanized and eyes enucleated. Sections (5 m thick) were obtained and stained using H&E. Eyes from SD rats without any injection were used as control. Magnification of the eye is 4×. Magnification is at 20× and shows the site of injection in A. A similar area was magnified in the control eye (B).

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Discussion

This is the first study to demonstrate sustained delivery of a therapeutic monoclonal antibody from a polycaprolactone dimethacrylate (PCM) and hydroxyethyl methacrylate (HEMA) gel (Figure 6.10). We have shown that 1) the gel sustained the in vitro release of bevacizumab for 4 months; 2) the gel maintained the monomeric form and activity of bevacizumab with little aggregation/degradation; and 3) bevacizumab entrapped in the light activated, in situ forming gel sustained in vivo delivery of bevacizumab following suprachoroidal injection in SD rats.

Figure 6.10 : Schematic representation of sustained delivery of bevacizumab from a polycaprolactone dimethacrylate (PCM) and hydroxyethyl methacrylate (HEMA) gel following in situ cross linking 365 nm UV light.

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Gel sustained in vitro bevacizumab release and activity for four months

Sustained release of bevacizumab from PCM and HEMA gel was investigated using an ELISA method (enzyme-linked immunosorbent assay). Since the ELISA assay measures the active form of bevacizumab, any inactivated bevacizumab in the release samples was not quantified by this assay. An increase in cross-linking duration resulted in a decrease in burst release from 81 to 21%, with the 10 minute cross-linking resulting in the lowest burst release (Figure 6.3). Following burst release, bevacizumab release was sustained for up to 4 months. It is generally desired that the burst release is minimized to ensure enough drug is retained for sustained release (Olsen, Feng et al. 2011). However, the burst release may serve as the loading dose to obtain immediate effects, while the slow release phase serves as the maintenance dose to sustain drug effects. Bevacizumab is expected to neutralize VEGF (Ferrara, Hillan et al. 2005) in tissues such as the choroid and retinal pigment epithelial (RPE) cells (Kliffen, Sharma et al. 1997; Marneros, Fan et al. 2005; Bhutto, McLeod et al. 2006) that overexpress VEGF in ocular diseases. The reported IC 50 of bevacizumab for inhibition of VEGF165 is 22 ng/ml (0.15nM) (Wang,

Fei et al. 2004). The second phase of release (following burst) provided sustained bevacizumab levels of > 1 µg/ml in the release medium for up to 4 months. Therefore, persistent beneficial effects are anticipated with such a gel formulation. Since the bevacizumab ELISA we used is a VEGF binding assay, it only measures bevacizumab that retains its ability to bind to VEGF. Thus, our in vitro release indicates that the gel sustains the release and activity of bevacizumab up to 4 months.

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Gel maintained the monomeric form and activity of bevacizumab with little degradation

Bevacizumab potentially can be rendered unstable during the preparation of the gel or during its residence and release. To obtain insight into the physical and conformational stability of gel-released bevacizumab, size exclusion chromatography and far UV circular dichroism were employed.

Size exclusion chromatography of released bevacizumab demonstrated the presence of high molecular weight aggregates (Figure 6.4) that likely correspond to dimers or trimers of bevacizumab (Kahook, Liu et al. 2010; Liu, Ammar et al. 2011).

Aggregates were observed in both control and release study samples. Furthermore, we observed the emergence of fragments during the release study. The appearance of fragments may be due to the degradation of the protein at the incubation temperature or as a consequence of the formulation ingredients, the UV light and/or photoinitator used in our study.

Bevacizumab had a distinct ellipticity at ~218 nm in the CD spectra (Figure 6.5).

In our study, bevacizumab maintained its secondary structural features after entrapment and release from the gel. Furthermore, the CD spectrum of released bevacizumab closely resembled that of control bevacizumab, indicating lack of structural changes.

Gel retained bevacizumab levels in the suprachoroidal space for over 60 days in

SD rats

An unmet need in the therapy of CNV is the development of slow release systems to sustain protein delivery in order to avoid the lifelong burden of monthly injections of anti-VEGF protein drugs. Since macromolecules such as bevacizumab clear rapidly from

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the suprachoroidal space (Olsen, Feng et al. 2011), we developed a photo-responsive delivery system that forms a gel in suprachoroidal space for localized, sustained delivery to diseased tissue. We assessed the injectability and retention of our release system in the suprachoroidal space of excised rabbit eyes. Injectability is of importance for ocular delivery systems, as large bore needles such as 20 or 25 gauge have higher risks of endophthalmitis (Kunimoto and Kaiser 2007) and hypotony (Acar, Kapran et al. 2008) when used for ocular delivery by intravitreal injections. Furthermore, suprachoroidal injection using a large bore needle might cause hemorrhage (Low, Ng et al. 2009). If a drug formulation can be delivered through a 30 gauge or smaller gauge needle, the system is expected to be safer than the larger gauge needle (Marsh 2011). During our experiments, we observed that our gel forming monomers were easily injected into the suprachoroidal space of excised rabbit eyes using a 30 gauge needle and later, these gels were cross-linked in-situ for sustained delivery. We also observed that after cross-linking for 10 minutes, Alexa Fluor ® 488 dye conjugated bevacizumab was retained at the site of injection for at least 48 h in the excised rabbit eye. However, an initial rapid decrease in signal was observed, which might correspond to dilution of the gel or burst release.

Beyond the initial decrease, a near constant level was maintained in the suprachoroidal space. In agreement with our in vitro studies, we found that the gel system encapsulating bevacizumab was effective in sustaining the delivery of Alexa Fluor ® 488 dye conjugated bevacizumab during a 60 day study in SD rats. To our knowledge this is the first report demonstrating the in vivo sustained delivery of bevacizumab for 60 days.

One limitation of our system was the use of a photoinitator that breaks down in to a benzoyl free radical. Even though the free radical is immediately consumed in the cross

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linking process, it can induce some toxicity. Excessive free radicals are known to cause oxidative stress, and damage lipid, protein and DNA in the eye (Qu, Wang et al. 2010).

Conclusions

A photo-responsive biodegradable gel was successfully synthesized and evaluated as a sustained release system for bevacizumab. The gel sustains the release of the entire loaded protein without affecting the stability of the protein. Further, our study demonstrates the significant potential of gels to sustain in vivo suprachoroidal delivery of macromolecules. Thus, the novel gel formulation surpasses the current state of art for sustained release of proteins in the eye.

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CHAPTER VII

SUMMARY AND FUTURE DIRECTIONS

Vascular endothelial growth factor (VEGF), a pro- angiogenic molecule, has been identified as a culprit for promoting angiogenesis in choroidal neovascualrization (CNV) and other angiogenic disorders(Hoeben, Landuyt et al. 2004). With the identification of

VEGF as a primary pro-angiogenic factor, anti-VEGF therapies became useful options for the treatment of angiogenesis. Most of the currently used anti-VEGF modalities are

VEGF binding antibodies or antibody fragments, which act extracellularly by binding the secreted VEGF and interfering with extracellular VEGF signaling, without inhibiting intracellular VEGF signaling. However, the anti-VEGF therapies are not effective in ~

60% of the patients (Rosenfeld, Brown et al. 2006). Furthermore, recent studies have suggested that angiogenic factors other than VEGF such as acidic and basic fibroblast growth factor (Amin, Puklin et al. 1994), cyclooxygenase-2 (COX2) (Castro, Lutz et al.

2004), and platelet-derived growth factor (PDGF) (Kudelka, Grossniklaus et al. 2013) also play a role in angiogenesis during CNV. Thus, other factors can cause angiogenesis and may easily take over the role of VEGF following anti-VEGF therapy. Tumor- associated fibroblasts, a major component of the angiogenic stroma from tumors resistant to anti-VEGF therapy, promote tumor growth by up regulating the expression of pro- angiogenic genes, including PDGF, and COX2 (Crawford, Kasman et al. 2009). Placental growth factor (PlGF) has been shown to be upregulated in metastatic colorectal cancer patients being treated with bevacizumab and induced resistance against bevacizumab 131

(Lieu, Tran et al. 2013). Further, an analysis of human breast cancer biopsies exposed that late-stage breast cancers expressed a lot of non-VEGF, pro-angiogenic factors such as FGF2 (Relf, LeJeune et al. 1997). Recently, Chung et al. discovered that an interleukin-17 mediated paracrine network promotes tumor resistance to anti-angiogenic therapies (Chung, Wu et al. 2013). Thus, it is important that newer therapies are developed and made available for the majority of patients who do not respond to anti-

VEGF therapies or develop resistance to these therapies.

Endogenous anti-angiogenic molecules such as tumstatin may be a better choice over the current anti-VEGF therapies because tumstatin inhibits angiogenesis by inhibiting multiple pathways such FAK/Akt/NF κB (nuclear transcription factor-kappa B) pathways, leading to decreased tumor angiogenesis and tumor growth in a α3β1 integrin dependent manner (Boosani, Mannam et al. 2007), unlike anti-VEGF antibodies that inhibit only VEGF. Tumstatin has also been shown to inhibit expression of angiogenic molecules such as VEGF and basic fibroblast growth factor (bFGF) (Boosani, Mannam et al. 2007). It is the decrease in expression of endogenous inhibitors such as tumstatin and eventually the shifting of the balance between pro- and anti-angiogenic stimuli that leads towards progression of angiogenesis (Sudhakar and Boosani 2008). It has been reported that endogenous inhibitors such as tumstatin have decreased production by 18- fold in human lungs suffering from asthma (Burgess, Boustany et al. 2010). We hypothesized that using an endogenous inhibitor such as tumstatin can be a novel therapeutic in comparison to the existing anti-VEGF therapies available.

A drawback of using recombinant tumstatin was its high dose requirement.

Tumstatin was used at a dose of 1.4 mg/kg every day for treating cancer in C57/BL6

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mice (Eikesdal, Sugimoto et al. 2008). In the same study, a separate group of mice were given bevacizumab once a week at a 7-fold lower dose to attain a similar effect.

Therefore, more efficacious derivatives of tumstatin would be beneficial in facilitating its clinical use. Towards this goal, we developed and evaluated a fusion protein, transferrin- tumstatin, to enhance efficacy of tumstatin protein. Further, we evaluated the safety and pharmacokinetics of transferrin-tumstatin in rabbits.

Convenient and safe delivery of therapeutic agents to the posterior ocular tissues remains a major challenge. Several permeability barriers exist in the eye, limiting delivery of therapeutics to the posterior ocular tissues. Following topical delivery, cornea and conjunctiva are the primary barriers because of epithelial barriers in these tissues that limit drug absorption due to the presence of tight junctions. Conjuncitval and episcleral blood supply are the subsequent barriers to delivery which can potentially clear the drug.

In addition, lymphatic clearance from the conjunctiva also limits drug delivery. It has been shown that macromolecules such as IgG are cleared by lymphatic circulation (Lee,

He et al. 2010). For the drug to enter the posterior tissues from the systemic circulation, it has to cross the retinal blood vessels, with a tight monolayer of endothelial cells (inner blood-retinal-barrier). Due to the presence of such formidable barriers, eye drops and systemic doses do not efficiently deliver a drug to the back of the eye tissues. Therefore, intravitreal injections are the norm to deliver drugs to the back of the eye (Kompella,

Kadam et al. 2010) and the currently approved anti-VEGF agents for the treatment of

AMD are administered intravitreally. However, intravitreally injected therapeutics also face barriers to reach the target site of choroid in treating CNV. The drug has to cross retina followed by retinal pigment epithelium, a monolayer of cells with tight junctions

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(outer blood-retinal-barrier). Further, intravitreal injections can cause retinal detachment and potential vision loss. Thus, development of less invasive approaches that are effective in local drug delivery to the target choroid tissue needs further investigation. Therefore, we developed the suprachoroidal route in rats, which offers a relatively safer and localized route of drug administration to the choroid-retina region of the eye.

An increasing number of recombinant proteins are being developed and commercialized nowadays. However, due to the rapid clearance of proteins therapeutics from the blood, monthly injections are necessary to maintain therapeutically effective concentrations. Therefore, sustained delivery systems of therapeutic proteins are attractive and would help reduce the frequency of injections and adverse side effects.

During the past few years, lot of effort has been put into developing sustained delivery systems such as polymeric particles for protein therapeutics (Vila, Sanchez et al. 2002).

However, the delivery systems are plagued with drawbacks such as protein degradation

(Bittner, Morlock et al. 1998), (Zambaux, Bonneaux et al. 1999) and protein aggregation

(Panyam, Dali et al. 2003).

Thus, the overall objective of this project was to express, characterize, and evaluate the efficacy of transferrin-tumstatin in vitro and in a disease model,(1) to assess the safety and pharmacokinetics of transferrin-tumstatin in a rabbit model following single intravitreal injection, (3) to develop suprachoroidal route of delivery in rats and evaluate the pharmacokinetics of sodium fluorescein by non-invasive ocular fluorophotometry, and to develop a sustained release system for suprachoroidal delivery of protein therapeutics using bevacizumab as a model protein.

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Specific aim 1

To design, express, purify, characterize, and evaluate the efficacy of transferrin- tumstatin in vitro and in vivo in a disease model.

Summary

We hypothesized that transferrin-tumstatin can be expressed and purified as a fusion protein. Further, we hypothesized that transferrin-tumstatin will have superior in vitro and in vivo activity when compared to bevacizumab, an anti-VEGF monoclonal antibody, and tumstatin, the parent protein. To test our hypothesis, we expressed transferrin-tumstatin in a mammalian expression system of ARPE cells and characterized it using SDS-PAGE, circular dichroism, fluorescence spectroscopy, size exclusion chromatography, and western blotting. We evaluated the efficacy of transferrin-tumstatin, bevacizumab, and tumstatin in a rat model of choroidal neovascularization. Our study demonstrated that transferrin-tumstatin is superior to bevacizumab and tumstatin in inhibiting choroid endothelial cell proliferation, tube formation, and migration. The in vivo efficacy of transferrin-tumstatin was superior to bevacizumab and tumstatin in inhibiting CNV in a rat model.

Significant findings

We developed a new anti-angiogenic fusion protein, transferrin-tumstatin, which inhibits proliferation of endothelial cells. The fusion protein showed better in vitro and in vivo efficacy when compared to bevacizumab and the parent protein tumstatin. The fusion of transferrin to tumstatin resulted in the cellular uptake of the fusion protein, whereas tumstatin uptake was not evident. The fusion protein exhibited a preferential basolateral secretion following exposure to retinal pigment epithelial cells. In silico

135

modeling demonstrated the superior binding of transferrin-tumstatin to αvβ3 integrin receptor in comparison to tumstatin alone.

Future directions

Based on the above results, investigations can be performed to test the following hypotheses.

Mechanisms for superior efficacy of transferrin-tumstatin:We hypothesize that transferrin-tumstatin protein, once internalized, increases the cytoplasmic content of

RGD containing proteins such as tumstatin and fibronectin thereby causing apoptosis

(Figure 7.1). Proteins similar to tumstatin, such as endostatin and angiostatin have been shown to form complexes with RGD containing plasma adhesion proteins such as fibronectin and vitronectin (Yi, Sakai et al. 2003). Accumulation of RGD containing proteins in the cytoplasm has been reported to initiate specific caspse-3 dependent apoptosis. Thus, internalization of anti-angiogenic factor/adhesion protein complexes

(e.g., tumstatin/fibronectin) could provide a supply of cytoplasmic RGD-containing proteins in the cytoplasm, thereby inducing apoptosis. This possibility is negligible for tumstatin as it did not show any signs of internalization in our studies. The following questions need to be answered in order to clarify this hypothesis.

1. Will Tf-T protein cause internalization of fibronectin and activation of caspases?

2. Which caspases are activated?

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Figure 7.1: Hypothetical mechanism of superior efficacy of transferrin-tumstatin over tumstatin

Identification and development of peptide fragments responsible for the basolateral secretion of transferrin-tumstatin: We analyzed the sequences of various secretory proteins and identified fragments of transferrin that can potentially cause basolateral secretion. Our analysis of the peptide sequence of various basolaterally secreted proteins such as interleukin 6, interleukin 8 (Holtkamp, Van Rossem et al.

1998), and vascular endothelial growth factor A (VEGF-A) (Blaauwgeers, Holtkamp et al. 1999) led us to identify a surprising similarity in the N terminal amino acids of such proteins. These proteins are secreted on the basolateral side and have an abundance of

137

leucine amino acid. Furthermore, dileucines (LL) are also present near the N terminal of these proteins. The following questions can be addressed in future.

1. Can we identify and prepare peptides from transferrin that are responsible for

basolateral secretion?

2. Do the peptides exhibit basolateral secretion of transferrin peptide-tumstatin

fusion proteins?

Specific aim 2

To determine the pharmacokinetics and safety of transferrin-tumstatin in a rabbit model following single intravitreal injection.

Summary

We hypothesized that transferrin-tumstatin will be cleared from the vitreous with a half-life of a few days from the vitreous following intravitreal injection. We also hypothesized that transferrin-tumstatin will be safe following intravitreal injection. To test our hypotheses, we intravitreally injected Alexa 488 conjugated transferrin-tumstatin in rabbit eyes. We monitored the clearance of Alexa conjugated transferrin-tumstatin from the eye by non-invasive ocular fluorophotometry. We assessed the safety of the injection by monitoring the rabbit for any ocular side effects using ophthalmic and physical examination. We found that transferrin-tumstatin was eliminated from the vitreous with a half life of 1.7 days. Physical examination indicated no drug related changes in the body weight, heart rate, or temperature of rabbits. Ocular examination was performed using fundus camera and slit lamp imaging and it was observed that vitreal floater-like appearance in one out four rabbits (possibly due to an air bubble injection) was seen at the end of day 1 following injection of Tf-T. However, the floater like object 138

could not be visualized by day 2. No cells or flare was observed in aqueous humor at any time point.

Significant findings

We observed that transferrin-tumstatin was cleared from the vitreous with a half- life of 1.7 days. Transient hyperemia was observed. Fundus imaging of the back of the eye confirmed the lack of any toxic effect to the back of the eye following intravitreal injection of transferrin-tumstatin.

Future directions

The pharmacokinetic and safety study provided us with preliminary data indicating safety of the protein following intravitreal injection. However, further studies need to be performed to confirm the long term safety of the protein. Questions that still need to be answered include:

1. Is the protein safe following repeated dosing in rabbits?

2. Is there any cross activity of transferrin-tumstatin in other tissues including brain,

heart, kidney, liver, lungs, spleen, stomach, and uterus?

3. What is the hemolytic potential and vitreal fluid compatibility of transferrin-

tumstatin?

Specific aim 3

To demonstrate suprachoroidal injection in a rat model and compare the pharmacokinetics of suprachoroidal injection with intravitreal and posterior subconjunctival injections using noninvasive ocular fluorophotometry.

139

Summary

We hypothesized that suprachoroidal injections are feasible in rats and the extent and rate of delivery of sodium fluorescein to choroid-retina is highest following suprachoroidal delivery, when compared to intravitreal and posterior subconjunctival injections. In our study histological analysis indicated localization of India ink to the suprachoroidal space below sclera, following injection with 10 µl glass syringe fitted with a 34 gauge needle (1/2 inch long; with a 45° taper). Localization of India ink confirmed our suprachoroidal injection technique. Sodium fluorescein delivery to choroid retina region was in the order: suprachoroidal > intravitreal > posterior subconjunctival injection.

Significant findings

The rate and extent of delivery of sodium fluorescein to the choroid-retina region was the highest following suprachoroidal injection. In comparison to suprachoroidal delivery, intravitreal injection had 4-fold lower Cmax (ng/ml) and 2-fold lower area under the curve (AUC; (ng*min)/ml) indicating lower bioavailability to the choroid-retina region by intravitreal injections. However, following suprachoroidal injection, sodium fluorescein was cleared out within 6 hours from the choroid-retina region. This may be due the abundance of blood vessels in the choroid.

Future directions

Based on the above results, future investigations will try to answer the following questions:

1. Is suprachoroidal injection of macromolecules such as transferrin-tumstatin

possible in rats?

140

2. What is the rate and extent of delivery of transferrin-tumstatin in the

suprachoroidal space following suprachoroidal injection in rats?

3. What is the efficacy of transferrin-tumstatin in a rat CNV model following

suprachoroidal injection?

Specific aim 4

To develop an in situ forming gel to sustain the release of stable, active bevacizumab and assess the sustained in vivo delivery in rat eyes following in situ gel formation in the suprachoroidal space.

Summary

We hypothesized that an in situ forming cross-linked gel will sustain the delivery of bevacizumab in vivo and in vitro. We also hypothesized that the gel provides sustained release of monomeric and active protein and is not toxic to ocular tissues following in vivo delivery. Our study demonstrated that in situ forming gel can sustain the release of protein for more than 4 months in vitro and for 2 months in vivo in SD rats.

Bevacizumab released in vitro was in active form and had ~ 5% or less of fragmentation.

The in situ forming gel was also evaluated for in vitro and in vivo toxicity. For assessment of in vitro toxicity, ARPE-19 cells were incubated with the gel forming polymers (PCM, HEMA, and photoinitator) without cross-linking, and also following cross-linking for 3, 7, or 10 minutes. At the end of 24 hours, the toxicity was assessed using MTT reagent. Gel forming polymers and polymers cross-linked for 3 and 7 minutes showed > 20% cell death. Polymers cross-linked for 10 minutes showed ~ 5% cell death.

In vivo toxicity was assessed by injecting the gel forming polymers and cross-linking for

10 minutes. At the end of 40 days following injection, rat eyes were enucleated and 5 µm

141

cross sections were attained and stained with hematoxylin and eosin to assess any morphological changes.

Significant findings

We observed that the in situ forming gel sustained in vitro bevacizumab release and activity for four months. Gel maintained the monomeric form and activity of bevacizumab with little degradation. Furthermore, the in situ forming gel retained bevacizumab levels in the suprachoroidal space for over 60 days in SD rats. Following suprachoroidal injection the in situ gel system was found to be safe.

Future directions

Our preliminary study indicated that the in situ forming gel can be potentially useful for sustained release of protein therapeutics in the suprachoroidal region.

However, further work is needed to completely understand and improve the delivery system. Following studies will be useful to advance the delivery system.

1. What is the mechanism of release of bevacizumab from the polycaprolactone

dimethacrylate and hydroxyl ethyl methacrylate gel based delivery system?

2. Can we modulate the polymer composition to extend the duration of protein

release from the gel?

3. What was the reason for bevacizumab fragmentation? Was there any modification

(e.g., oxidation) in bevacizumab that led to the fragmentation of the protein?

4. How can we prevent the fragmentation of bevacizumab during long term in vivo

delivery?

142

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