**Meet the editor**

Dr Ying is an Assistant Professor of Ophthalmology, a faculty biostatistician in the Center of Preventive Ophthalmology and Biostatistics at the Department of Ophthalmology, and an Associate Scholar at the Department of Biostatistics, Perelman School of Medicine, University of Pennsylvania. He has more than 10 years of research experience in Age-related Macular Degeneration (AMD)

and is the senior biostatistician for several multi-center AMD clinical trials, including the Complications of AMD Prevention Trial (CAPT), and the Comparison of AMD Treatment Trials (CATT). He collaborates with ophthalmologists and vision scientists and has authored or co-authored more than 80 papers in peer-reviewed journals. Dr Ying received a MD in Preventive Medicine and a MPH in Toxicology, both from ZheJiang University in China, and a PhD in Biostatistics from the University of Pennsylvania in the United States.

Contents

**Preface IX** 

Suofu Qin

Chapter 3 **Bruch's Membrane:** 

Chapter 6 **Basic Research and** 

**Part 2 Clinical Research 121** 

Chapter 7 **Treatment of Neovascular** 

Chapter 8 **Re-Treatment Strategies for** 

Chapter 5 **Experimental Treatments for** 

**Part 1 Basic and Translational Research 1** 

Chapter 1 **Wet Age Related Macular Degeneration 3** 

Chapter 2 **Pathogenic Roles of Sterile Inflammation in** 

Robert F. Mullins and Elliott H. Sohn

Chapter 4 **Non-Enzymatic Post-Translational Modifications in** 

Yuichi Kaji, Tetsuro Oshika and Noriko Fujii

C. V. Regatieri, J. L. Dreyfuss and H. B. Nader

Giuseppe Lo Giudice and Alessandro Galan

**Age Related Macular Degeneration 123** 

Sengul Ozdek and Mehmet Cuneyt Ozmen

Ratimir Lazić and Nikica Gabrić

**Clinical Application of Drug Delivery Systems** 

Fardad Afshari, Chris Jacobs, James Fawcett and Keith Martin

**Etiology of Age-Related Macular Degeneration 25** 

**The Critical Boundary in Macular Degeneration 49** 

**Neovascular Age-Related Macular Degeneration 83** 

**the Development of Age-Related Macular Degeneration 73** 

**for the Treatment of Age-Related Macular Degeneration 99** 

**Neovascular AMD: When to Treat? When to Stop? 143** 

### Contents

#### **Preface XI**


	- **Part 2 Clinical Research 121**

X Contents



### Preface

In the past decade, great progress has been made in understanding the pathobiology and genetics of Age‐Related Macular Degeneration (AMD), and the effective therapies for this blinding disease have become available. These advancements have lead to the substantial change in the management of AMD patients. The online book *Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care* presents the most recent advances in basic research and clinical care of AMD. Different from other AMD books, this book aims to cover the new findings from basic and translational research on the biological and genetic mechanism of AMD, and the new interventions to prevent and treat this disease.

The book has a total of 15 chapters, grouped into two sections. Section one includes six chapters covering the basic and translational research of AMD. Section two includes nine chapters describing the clinical research and management of AMD. Each chapter has been contributed to by outstanding researchers or clinicians in the area of AMD. They present a very detailed review of new research and findings in the topic‐specific AMD area, and also provide direction for future research. The book is targeted at researchers and clinicians who are interested in learning about new advances in the understanding and treatment of AMD, and insights into future research of AMD.

We hope that this AMD book will provide the latest information to its readers. The large amount of information presented in this book will help clinicians to take best care of their AMD patients. Additionally, it will assist researchers in conducting further AMD research and, eventually, achieve the goal of finding effective and safe ways to prevent or treat AMD.

> **Gui‐Shuang Ying, PhD** Assistant Professor of Ophthalmology University of Pennsylvania, Perelman School of Medicine Philadelphia, PA USA

**Part 1** 

**Basic and Translational Research** 

## **Part 1**

**Basic and Translational Research** 

**1** 

*UK* 

*University of Cambridge,* 

**Wet Age Related Macular Degeneration** 

Fardad Afshari, Chris Jacobs, James Fawcett and Keith Martin

Age related macular degeneration (AMD) is the leading cause of blindness in the developed countries. Approximately 8 million people in America have AMD and the number of advanced AMD is likely to rise by 50% by year 2020 due to the projected increase in the number of elderly people (Friedman et al., 2004). AMD is a condition of significant morbidity in terms of both physical and mental health (Hassell et al 2006). The burden of this disease is multifaceted as both the individual and society bear a cost. The individual has a loss of independence and ability of self care, with a pressure on society to fulfil the need

In this review of AMD, we will explore the epidemiology of AMD, the criteria for diagnosis

AMD affects a large proportion of the elderly population. By applying the criteria of presence of macular drusen greater than 63 micrometres in diameter on fundus photography, up to 61% of adults over 60 years have some degree of AMD (Piermarocchi et al 2011). With a high estimated prevalence, it is important to understand the potential risk

A meta analysis of published data suggests that increasing age, current cigarette smoking, previous cataract surgery, and a family history of AMD show strong and consistent associations with late AMD. Risk factors with moderate and consistent associations were higher body mass index, history of cardiovascular disease, hypertension, and higher plasma fibrinogen. Risk factors with weaker and inconsistent associations were gender, ethnicity, diabetes, iris colour, history of cerebrovascular disease, and serum total and HDL

Direct associations between AMD and age, cataract, family history, alcohol consumption, the apolipoproteins A1 and B were also found in a 14 year follow up amongst a city populations (Buch et al 2005). In addition, recent data on human genome project have linked a complement H polymorphism Try402His on chromosome 1 to increased risk of AMD (Klein et al.,2005). Ala69ser polymorphism in the ARMS2 gene on chromosome 10 is yet another instance where genetic susceptibility for this condition has been established (Rivera et al., 2005). It has also been shown that ARMS2 polymorphism together with smoking, can

with particular focus on the pathophysiology and treatments of wet AMD.

cholesterol and triglyceride levels (Chakravarthy et al 2010).

**1. Introduction** 

**1.1 Epidemiology** 

factors for this condition.

for community and vision related support.

## **Wet Age Related Macular Degeneration**

Fardad Afshari, Chris Jacobs, James Fawcett and Keith Martin *University of Cambridge, UK* 

#### **1. Introduction**

Age related macular degeneration (AMD) is the leading cause of blindness in the developed countries. Approximately 8 million people in America have AMD and the number of advanced AMD is likely to rise by 50% by year 2020 due to the projected increase in the number of elderly people (Friedman et al., 2004). AMD is a condition of significant morbidity in terms of both physical and mental health (Hassell et al 2006). The burden of this disease is multifaceted as both the individual and society bear a cost. The individual has a loss of independence and ability of self care, with a pressure on society to fulfil the need for community and vision related support.

In this review of AMD, we will explore the epidemiology of AMD, the criteria for diagnosis with particular focus on the pathophysiology and treatments of wet AMD.

#### **1.1 Epidemiology**

AMD affects a large proportion of the elderly population. By applying the criteria of presence of macular drusen greater than 63 micrometres in diameter on fundus photography, up to 61% of adults over 60 years have some degree of AMD (Piermarocchi et al 2011). With a high estimated prevalence, it is important to understand the potential risk factors for this condition.

A meta analysis of published data suggests that increasing age, current cigarette smoking, previous cataract surgery, and a family history of AMD show strong and consistent associations with late AMD. Risk factors with moderate and consistent associations were higher body mass index, history of cardiovascular disease, hypertension, and higher plasma fibrinogen. Risk factors with weaker and inconsistent associations were gender, ethnicity, diabetes, iris colour, history of cerebrovascular disease, and serum total and HDL cholesterol and triglyceride levels (Chakravarthy et al 2010).

Direct associations between AMD and age, cataract, family history, alcohol consumption, the apolipoproteins A1 and B were also found in a 14 year follow up amongst a city populations (Buch et al 2005). In addition, recent data on human genome project have linked a complement H polymorphism Try402His on chromosome 1 to increased risk of AMD (Klein et al.,2005). Ala69ser polymorphism in the ARMS2 gene on chromosome 10 is yet another instance where genetic susceptibility for this condition has been established (Rivera et al., 2005). It has also been shown that ARMS2 polymorphism together with smoking, can

Wet Age Related Macular Degeneration 5

To classify AMD, multiple ophthalmological tools have proven to be useful including dilated indirect ophthalmoscopy, stereoscopic fundus photography, amsler grid testing, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). Of the mentioned techniques available, FFA is of great importance as it allows differentiation between neovascularisation attributable to AMD and that caused by other conditions. The use of FFA has enabled sub-classification of wet AMD according to the appearance of the lesions and the location of choroidal neovascularisation in relation to the fovea. The appearance can be described as classic or occult, which is according to the defined features of the membrane at early and late phases. The location can be extrafoveal (choroidal neovascularisation greater than 200um from the foveal avascular zone), juxtafoveal (choriodal neovascularisation is closer than 200um from the fovealavascualr zone) and sub-foveal (originating or extension of choroidal neovascularisation to the centre of the avascular zone). OCT provides a cross sectional image of the macula and identifies retinal pigment detachment, fluid accumulation and vitero-macular attachments. OCT has become an important tool in the monitoring

In this section we will explore the clinical presentation and the current pathophysiological

Clinically, AMD presents with visual loss of varying severity. Early in the course of disease, patients can present with very mild symptoms or be completely asymptomatic. Some patients, however, do experience a loss of contrast sensitivity, blurred vision and scotomas as the disease progresses to the intermediate stage (Jager et al., 2008). Other visual abnormalities associated with AMD include metamophopsia(distortion of straight lines), disparity of image size, macropisa and micropsia, hyperopic refractive shift with associated anisometriopia, light glare, floaters, photopsia (Schmidt-Erfurth et al 2004). However, neovascular or wet AMD, unlike the dry subtype, can have a sudden onset of presentation due to subretinal haemorrhages and exudates leading to retinal detachment and a acute visual loss (Jager et al., 2008). Although wet AMD is only responsible for 15% of the total AMD, it is responsible for

Various theories and models have been proposed to explain the pathophysiology of AMD with multiple factors contributing to the final outcome. Most models proposed focus either

Retinal pigment epithelial (RPE) cells, form a single layer of cells overlying Bruch's membrane with photoreceptors located anterior to RPE layer. RPE cells play a very complex role in preserving photoreceptors and their function. One of their major functions is to remove the shed outer segments of the photoreceptors by phagocytosis (Chang and Finnemann, 2007;Finnemann and Silverstein, 2001). It has been shown that failure of this process will result in build up of debris between the retinal layer and the Bruch's membrane

on the Bruch's membrane or on the retinal pigmented cells overlying this membrane.

more than 80% of AMD-related severe visual loss and blindness (Fine et al., 1986).

**2.2 Pathophysiological models for AMD development** 

leading to retinal degeneration (Nandrot et al., 2004).

progression of wet AMD especially in light of new therapeutic possibilities.

**2. Pathophysiology of wet AMD** 

**2.1 Clinical presentation of wet AMD** 

mechanism underlying the development of AMD.

synergistically increase the risk of developing AMD (Schmidt et al., 2006). Therefore it is evident that AMD is a result of interplay of genetic and environmental factors leading to the final pathology.

Better understanding of risk factors can help to identify individuals at high risk for wet AMD who may benefit from early intervention with existing or novel therapies. Using visual acuity as an outcome measure, visual prognosis is more favourable in patients with early intervention (Wong et al 2008).

#### **1.2 Classification of AMD and diagnosis**

AMD is characterized by the deposition of polymorphous material between the retinal pigmented epithelium and Bruch's membrane (Jager et al., 2008). These depositions are named Drusen. Drusen are categorised by sizes as, small(<63μm), medium (63-124 μm) and large (>124μm) (Bird et al., 1995). They are also considered as hard or soft depending on the appearance of their margins on opthalmological examination. While hard drusens have clearly defined margins, soft ones have less defined and fluid margins (Bird et al., 1995).

Classically the condition is divided in to two main subtypes; dry/non exudative and wet/exudative. The Age-related Eye Disease Study (AREDS) fundus photographic severity scale is one of the main classification systems used for this condition (Sallo et al 2009):

#### **No AMD (AREDS category 1)**

No or a few small (<63 micrometres in diameter) drusen.

#### **Early AMD (AREDS category 2)**

Many small drusen or a few intermediate-sized (63-124 micrometres in diameter) drusen, or macular pigmentary changes.

#### **Intermediate AMD (AREDS category 3)**

Extensive intermediate drusen or at least one large (≥125 micrometres) drusen, or geographic atrophy not involving the foveal centre.

#### **Advanced AMD (AREDS category 4)**

Geographic atrophy involving the foveal centre (atrophic, or dry AMD)

Choroidal neovascularisation (wet AMD) or evidence for neovascular maculopathy (subretinal haemorrhage, serous retinal or retinal pigment epithelium detachments, lipid exudates, or fibrovascular scar).

Wet AMD results from the abnormal growth of blood vessels from the choriocapillaris (choroidal neovascularisation), through Bruch's membrane. The fragility of the blood vessels and inflammatory processes lead to subretinal haemorrhages and fibrovascular scarring. This process can occur de novo or as a progression of dry AMD.

As with many classification systems, there is variability in AMD grading between clinicians. Therefore although such scales are important for accurate follow up of AMD progression, care is needed in their interpretation.

synergistically increase the risk of developing AMD (Schmidt et al., 2006). Therefore it is evident that AMD is a result of interplay of genetic and environmental factors leading to the

Better understanding of risk factors can help to identify individuals at high risk for wet AMD who may benefit from early intervention with existing or novel therapies. Using visual acuity as an outcome measure, visual prognosis is more favourable in patients with

AMD is characterized by the deposition of polymorphous material between the retinal pigmented epithelium and Bruch's membrane (Jager et al., 2008). These depositions are named Drusen. Drusen are categorised by sizes as, small(<63μm), medium (63-124 μm) and large (>124μm) (Bird et al., 1995). They are also considered as hard or soft depending on the appearance of their margins on opthalmological examination. While hard drusens have clearly defined margins, soft ones have less defined and fluid margins (Bird et al., 1995).

Classically the condition is divided in to two main subtypes; dry/non exudative and wet/exudative. The Age-related Eye Disease Study (AREDS) fundus photographic severity scale is one of the main classification systems used for this condition (Sallo et al 2009):

Many small drusen or a few intermediate-sized (63-124 micrometres in diameter) drusen, or

Extensive intermediate drusen or at least one large (≥125 micrometres) drusen, or

Choroidal neovascularisation (wet AMD) or evidence for neovascular maculopathy (subretinal haemorrhage, serous retinal or retinal pigment epithelium detachments, lipid

Wet AMD results from the abnormal growth of blood vessels from the choriocapillaris (choroidal neovascularisation), through Bruch's membrane. The fragility of the blood vessels and inflammatory processes lead to subretinal haemorrhages and fibrovascular scarring.

As with many classification systems, there is variability in AMD grading between clinicians. Therefore although such scales are important for accurate follow up of AMD progression,

final pathology.

early intervention (Wong et al 2008).

**No AMD (AREDS category 1)** 

**Early AMD (AREDS category 2)** 

**Intermediate AMD (AREDS category 3)** 

**Advanced AMD (AREDS category 4)** 

exudates, or fibrovascular scar).

care is needed in their interpretation.

geographic atrophy not involving the foveal centre.

Geographic atrophy involving the foveal centre (atrophic, or dry AMD)

This process can occur de novo or as a progression of dry AMD.

macular pigmentary changes.

No or a few small (<63 micrometres in diameter) drusen.

**1.2 Classification of AMD and diagnosis** 

To classify AMD, multiple ophthalmological tools have proven to be useful including dilated indirect ophthalmoscopy, stereoscopic fundus photography, amsler grid testing, fundus fluorescein angiography (FFA) and optical coherence tomography (OCT). Of the mentioned techniques available, FFA is of great importance as it allows differentiation between neovascularisation attributable to AMD and that caused by other conditions. The use of FFA has enabled sub-classification of wet AMD according to the appearance of the lesions and the location of choroidal neovascularisation in relation to the fovea. The appearance can be described as classic or occult, which is according to the defined features of the membrane at early and late phases. The location can be extrafoveal (choroidal neovascularisation greater than 200um from the foveal avascular zone), juxtafoveal (choriodal neovascularisation is closer than 200um from the fovealavascualr zone) and sub-foveal (originating or extension of choroidal neovascularisation to the centre of the avascular zone). OCT provides a cross sectional image of the macula and identifies retinal pigment detachment, fluid accumulation and vitero-macular attachments. OCT has become an important tool in the monitoring progression of wet AMD especially in light of new therapeutic possibilities.

#### **2. Pathophysiology of wet AMD**

In this section we will explore the clinical presentation and the current pathophysiological mechanism underlying the development of AMD.

#### **2.1 Clinical presentation of wet AMD**

Clinically, AMD presents with visual loss of varying severity. Early in the course of disease, patients can present with very mild symptoms or be completely asymptomatic. Some patients, however, do experience a loss of contrast sensitivity, blurred vision and scotomas as the disease progresses to the intermediate stage (Jager et al., 2008). Other visual abnormalities associated with AMD include metamophopsia(distortion of straight lines), disparity of image size, macropisa and micropsia, hyperopic refractive shift with associated anisometriopia, light glare, floaters, photopsia (Schmidt-Erfurth et al 2004). However, neovascular or wet AMD, unlike the dry subtype, can have a sudden onset of presentation due to subretinal haemorrhages and exudates leading to retinal detachment and a acute visual loss (Jager et al., 2008). Although wet AMD is only responsible for 15% of the total AMD, it is responsible for more than 80% of AMD-related severe visual loss and blindness (Fine et al., 1986).

#### **2.2 Pathophysiological models for AMD development**

Various theories and models have been proposed to explain the pathophysiology of AMD with multiple factors contributing to the final outcome. Most models proposed focus either on the Bruch's membrane or on the retinal pigmented cells overlying this membrane.

Retinal pigment epithelial (RPE) cells, form a single layer of cells overlying Bruch's membrane with photoreceptors located anterior to RPE layer. RPE cells play a very complex role in preserving photoreceptors and their function. One of their major functions is to remove the shed outer segments of the photoreceptors by phagocytosis (Chang and Finnemann, 2007;Finnemann and Silverstein, 2001). It has been shown that failure of this process will result in build up of debris between the retinal layer and the Bruch's membrane leading to retinal degeneration (Nandrot et al., 2004).

Wet Age Related Macular Degeneration 7

Fig. 3. Fundus fluorescein angiography (FFA) image of corresponding eye affected by wet

Fig. 4. Optical coherence tomography (OCT) image of corresponding eye. Significant

AMD.

macular oedema is evident.

Fig. 1. Fundoscopic view- dry AMD. Note there is no neovascularisation evident.

Fig. 2. Fundoscopic view of wet AMD. Excessive neovascularisation in macular region.

Fig. 1. Fundoscopic view- dry AMD. Note there is no neovascularisation evident.

Fig. 2. Fundoscopic view of wet AMD. Excessive neovascularisation in macular region.

Fig. 3. Fundus fluorescein angiography (FFA) image of corresponding eye affected by wet AMD.

Fig. 4. Optical coherence tomography (OCT) image of corresponding eye. Significant macular oedema is evident.

Wet Age Related Macular Degeneration 9

suggested that the accumulation of large numbers of macular drusen is a necessity for the development of geographic atrophy and choroidal neovascularization characteristic of

Biochemical and immunohistological studies suggest drusen consist of immunoglobulins and components of the complement pathway (such as the C5b-C9 complex), acute phase response proteins raised in inflammation (CRP, amyloid P component and alpha1 antitrypsin), proteins that modulate the immune response (such as vitronectin, clusterin, apolipoprotein E, membrane cofactor protein and complement receptor1), major histocompatibility complex class 2 antigens, and HLA-DR and cluster differentiation antigens (Hageman et al., 1999; Johnson et al., 2000; Mullins et al.,2000; Sakaguchi et al., 2002; Zarbin, 2004). In addition, there are cellular components in drusen including RPE membrane debris, lipofuscin, melanin and choroidal dendritic cells (Ishibashi et al., 1986;

In support of this inflammatory theory, intravitreal injections of corticosteroids reduce the incidence of laser-induced CNVs in non human primates, possibly by reducing

It has been shown that with increasing age, oxidative damage in RPE cells also increases (Wallace et al., 1998). This is associated with a decrease in levels of antioxidant protective agents such as plasma glutathione, while oxidized glutathione levels increase. Also antioxidant vitamins, such as vitamin C and E, show a decline with increasing age (Rikans

In support of oxidation stress as one of the factors involved, accumulation of lipofuscin has been observed in aging eyes. Lipofuscins are derivatives of vitamin A metabolites (Katz et al., 1994). It has been shown that in the first decade of life, they only constitute 1% of the cytoplasmic volume of RPE cells where as this is increased to 19% of cytoplasmic volume in

*In vitro* studies suggest that RPE lipofuscin is a photo-inducible generator of reactive oxygen species. Lipofuscin granules are continuously exposed to visible light and to high oxygen tension, which causes the production of reactive oxygen species and oxidative damage to

RPE lipofuscin accumulation can ultimately lead to the disruption of lysosomal integrity, induce lipid peroxidation, reduce the phagocytic capacity of RPE cells and ultimately lead to loss of RPE cells (Boulton et al., 1993; De La Paz and Anderson, 1992; Sundelin and Nilsson,

Consistent with the oxidative stress model, clinical studies on the use of antioxidants has shown that in patients with extensive intermediate drusen, supplementation with antioxidant vitamins and minerals reduces the risk of developing advanced AMD from 28%

With aging, various changes can happen to the extracellular matrix deposited within the Bruch's membrane. It has been shown that there is a decline of laminin, fibronectin and type

advanced AMD (Harman, 1956; Wallace, 1999).

Killingsworth, 1987; Mullins et al., 2000).

and Moore, 1988; Vandewoude and Vandewoude, 1987).

the elderly (De La Paz and Anderson, 1992; Feeney-Burns et al., 1984).

RPE cells (Wassell et al., 1999; Winkler et al.,1999; Zarbin, 2004).

to 20% (Age related eye disease study research group, 2001).

inflammation (Ishibashi et al., 1985).

**2.2.2 Oxidative stress** 

2001; Zarbin, 2004).

**2.2.3 Abnormal ECM production** 

In AMD, various abnormalities in the Bruch's membrane have been shown to lead to the disruption of RPE function (Sun et al., 2007), and this in turn can lead to the disruption of photoreceptor function and their loss. Therefore, Bruch's membrane has been the focus of great deal of AMD research.

To understand the pathophysiology of AMD, it is necessary to understand the basic normal structure of Bruch's membrane. Bruch's membrane is a penta-laminar structure, composed of RPE basement membrane, inner collagenous layer, elastin lamina, outer collagenous layer and choriocapillary basement membrane (Zarbin et al 2003). Each layer has a different composition of extracellular ligands, capable of interacting with integrins on the RPE cells. The top layer of Bruch's membrane (the RPE basement membrane) is of great importance as it contains an important extracellular matrix called laminin (Das et al., 1990; Zarbin,2003; Pauleikhoff et al., 1990) necessary for RPE adhesion and attachment.

Over the years, molecular analysis of Bruch's membrane has lead to the identification of composition of each layer as summarized in the table below (Das et al., 1990; Zarbin, 2003; Pauleikhoff et al., 1990).


Table 1. Matrix components of different layers of Bruch's membrane.

Each layer of Bruch's membrane is composed of mixture of proteoglycans and adhesive ligands. Adhesive ligands interact with integrins on the surface of RPE cells. Different subunits of integrins interact with different class of ligands. RPE cells attachment to Bruch's membrane is largely dependent on integrin's ability to anchor the cell to the membrane firmly. Pathological states affecting the membrane or RPE cells therefore, may disrupt this important interaction leading to loss of adhesion and death of RPE cells.

A large number of hypotheses have existed regarding pathological processes involved in AMD. Overall, the pathological mechanisms proposed in AMD can be divided into 4 categories of inflammation, oxidative stress, abnormal ECM production, formation of CNVs and neovascularisation (Zarbin, 2004). These various components can happen either sequentially or they can occur simultaneously, leading to the final outcome seen in AMD (Zarbin, 2004).

#### **2.2.1 The inflammation component**

Although drusen formation is one of the hallmarks of AMD, controversy exists as to whether they are directly involved in the pathology of AMD. Drusen can be found in non-AMD patient eyes incidentally associated with aging (Zarbin, 2004). However, others have

In AMD, various abnormalities in the Bruch's membrane have been shown to lead to the disruption of RPE function (Sun et al., 2007), and this in turn can lead to the disruption of photoreceptor function and their loss. Therefore, Bruch's membrane has been the focus of

To understand the pathophysiology of AMD, it is necessary to understand the basic normal structure of Bruch's membrane. Bruch's membrane is a penta-laminar structure, composed of RPE basement membrane, inner collagenous layer, elastin lamina, outer collagenous layer and choriocapillary basement membrane (Zarbin et al 2003). Each layer has a different composition of extracellular ligands, capable of interacting with integrins on the RPE cells. The top layer of Bruch's membrane (the RPE basement membrane) is of great importance as it contains an important extracellular matrix called laminin (Das et al., 1990; Zarbin,2003;

Over the years, molecular analysis of Bruch's membrane has lead to the identification of composition of each layer as summarized in the table below (Das et al., 1990; Zarbin, 2003;

Each layer of Bruch's membrane is composed of mixture of proteoglycans and adhesive ligands. Adhesive ligands interact with integrins on the surface of RPE cells. Different subunits of integrins interact with different class of ligands. RPE cells attachment to Bruch's membrane is largely dependent on integrin's ability to anchor the cell to the membrane firmly. Pathological states affecting the membrane or RPE cells therefore, may disrupt this

A large number of hypotheses have existed regarding pathological processes involved in AMD. Overall, the pathological mechanisms proposed in AMD can be divided into 4 categories of inflammation, oxidative stress, abnormal ECM production, formation of CNVs and neovascularisation (Zarbin, 2004). These various components can happen either sequentially or

Although drusen formation is one of the hallmarks of AMD, controversy exists as to whether they are directly involved in the pathology of AMD. Drusen can be found in non-AMD patient eyes incidentally associated with aging (Zarbin, 2004). However, others have

they can occur simultaneously, leading to the final outcome seen in AMD (Zarbin, 2004).

sulphate

dermatan sulphate

Dermatan sulphate

laminin, heparan sulphate

Collagen IV, Collagen V, laminin, Heparan

Collagen I, Collagen III, Collagen V, fibronectin, Chondroitin sulphate,

Collagen I, Collagen III, Collagen V, fibronectin, Chondroitin sulphate,

Collagen IV, Collagen V, Collagen VI,

Pauleikhoff et al., 1990) necessary for RPE adhesion and attachment.

**Layer 3. Elastic lamina** Elastin, Collagen I, Fibronectin

Table 1. Matrix components of different layers of Bruch's membrane.

important interaction leading to loss of adhesion and death of RPE cells.

great deal of AMD research.

Pauleikhoff et al., 1990).

**membrane** 

**Layer 1. Basement membrane (Immediately underneath RPE layer)** 

**Layer 2. Inner collagenous layer** 

**Layer 4. Outer collagenous layer** 

**Layer 5. Choriocapillaries basement** 

**2.2.1 The inflammation component** 

suggested that the accumulation of large numbers of macular drusen is a necessity for the development of geographic atrophy and choroidal neovascularization characteristic of advanced AMD (Harman, 1956; Wallace, 1999).

Biochemical and immunohistological studies suggest drusen consist of immunoglobulins and components of the complement pathway (such as the C5b-C9 complex), acute phase response proteins raised in inflammation (CRP, amyloid P component and alpha1 antitrypsin), proteins that modulate the immune response (such as vitronectin, clusterin, apolipoprotein E, membrane cofactor protein and complement receptor1), major histocompatibility complex class 2 antigens, and HLA-DR and cluster differentiation antigens (Hageman et al., 1999; Johnson et al., 2000; Mullins et al.,2000; Sakaguchi et al., 2002; Zarbin, 2004). In addition, there are cellular components in drusen including RPE membrane debris, lipofuscin, melanin and choroidal dendritic cells (Ishibashi et al., 1986; Killingsworth, 1987; Mullins et al., 2000).

In support of this inflammatory theory, intravitreal injections of corticosteroids reduce the incidence of laser-induced CNVs in non human primates, possibly by reducing inflammation (Ishibashi et al., 1985).

#### **2.2.2 Oxidative stress**

It has been shown that with increasing age, oxidative damage in RPE cells also increases (Wallace et al., 1998). This is associated with a decrease in levels of antioxidant protective agents such as plasma glutathione, while oxidized glutathione levels increase. Also antioxidant vitamins, such as vitamin C and E, show a decline with increasing age (Rikans and Moore, 1988; Vandewoude and Vandewoude, 1987).

In support of oxidation stress as one of the factors involved, accumulation of lipofuscin has been observed in aging eyes. Lipofuscins are derivatives of vitamin A metabolites (Katz et al., 1994). It has been shown that in the first decade of life, they only constitute 1% of the cytoplasmic volume of RPE cells where as this is increased to 19% of cytoplasmic volume in the elderly (De La Paz and Anderson, 1992; Feeney-Burns et al., 1984).

*In vitro* studies suggest that RPE lipofuscin is a photo-inducible generator of reactive oxygen species. Lipofuscin granules are continuously exposed to visible light and to high oxygen tension, which causes the production of reactive oxygen species and oxidative damage to RPE cells (Wassell et al., 1999; Winkler et al.,1999; Zarbin, 2004).

RPE lipofuscin accumulation can ultimately lead to the disruption of lysosomal integrity, induce lipid peroxidation, reduce the phagocytic capacity of RPE cells and ultimately lead to loss of RPE cells (Boulton et al., 1993; De La Paz and Anderson, 1992; Sundelin and Nilsson, 2001; Zarbin, 2004).

Consistent with the oxidative stress model, clinical studies on the use of antioxidants has shown that in patients with extensive intermediate drusen, supplementation with antioxidant vitamins and minerals reduces the risk of developing advanced AMD from 28% to 20% (Age related eye disease study research group, 2001).

#### **2.2.3 Abnormal ECM production**

With aging, various changes can happen to the extracellular matrix deposited within the Bruch's membrane. It has been shown that there is a decline of laminin, fibronectin and type

Wet Age Related Macular Degeneration 11

to various factors in combination with the upregulation of VEGF, can synergistically lead to the choriocapillary CNVs penetrating the membrane and reaching the subretinal space

One of the molecules that has been studied extensively in our lab is a glycoprotein called tenascin C, known to be overexpressed in angiogenesis (Zagzag and Capo, 2002; Zagzaget al., 1996), neovascularisation and wound healing (Maseruka et al., 1997). Tenascin C deposition can occur in the Bruch's membrane in wet AMD on the basal side of RPE cells (Fasler-Kan et al., 2005) and in association with CNVs in the pathological Bruch's membrane (Nicolo et al., 2000). Tenascin C has been shown to prevent adhesion of RPE cells to extracellular matrix (Afshari et al 2010). Therefore accumulation of this molecule associated with CNV formation may play an important role in RPE loss from the Bruch's membrane

In summary, different pathological processes during aging and in AMD can lead to modifications in the Bruch's membrane which ultimately becomes a less supportive

*In vitro* models have allowed development of simplified systems to study processes involved in wet AMD. Most *in vitro* models have focused on the role of angiogenesis and

Tezel and Del priore first described methodology for accessing different layers of Bruch's membrane to allow *in vitro* assessment of RPE adhesion at different levels of Bruch's membrane. A combination of enzymatic treatment and mechanical techniques were used to expose each layer sequentially starting from the top basal lamina and moving to deeper structures. Using this technique, it was shown that deeper layers of Bruch's membrane are less supportive of RPE attachment (Del priore et al 1998; Tezel TH 1999 FEB; Tezel TH 1999 March). RPE cell adhesion to Bruch's membrane may play a detrimental role both in AMD

An alternative way of accessing Bruch's membrane used in our lab is the water lysis technique (Afshari et al 2010). In this method, eye globes are dissected out and separated from their muscle attachments. The anterior chamber is then dissected away leaving the posterior chamber and retina and Bruch's-choroid-sclera. Retinal layer is then carefully removed leaving the Bruch's-choroid-sclera trilaminar structure which can be subsequently exposed to water. Exposure to water leads to lysis of endogenous RPE cells. Lysed RPE cells are then flushed away from the surface of Bruch's membrane using a mini water jet. This procedure therefore results in formation of a denuded Bruch's membrane which can allow further experiments such as transplanting exogenous RPE cells to assess adhesion and migration of the transplanted cells (Afshari et al 2010). The advantages of this technique is that minimal treatment of the tissue is required with preservation of natural Bruch's membrane. In addition the preparation of the Bruch's membrane for adhesion and migration assay is a short procedure. Immunostaining of both frozen sections and electron microscopy of the membranes following water treatment have confirmed complete removal

(Schwesinger et al.,2001; Zarbin ,2004).

seen in AMD (Afshari et al 2010).

and following RPE transplantation.

environment for the RPE adhesion and function.

**3. Experimental models available for studying wet AMD 3.1** *In vitro* **and** *ex vivo* **models - Advantages vs disadvantages** 

isolation of Bruch's membrane to assess adhesion and survival of RPE cells.

IV collagen in the aging RPE basement membrane, particularly over the drusen (Pauleikhoff et al., 1999).

There is an age dependent increase in type I collagen within the Bruch's membrane, with an increase in the thickness of the membrane from 2 micrometres at birth, to up to 6 micrometres in the elderly ages (Ramrattan et al., 1994). During aging , the membrane glycosaminglycans in Bruch's membrane increase in size, and there is an increase in the heparan sulphate proteoglycan content of the membrane (Hewitt et al., 1989). Furthermore, glycation end products can accumulate within the Bruch's membrane with aging, trapping other macromolecules (King and Brownlee, 1996; Schmidt et al., 2000).

RPE cells themselves are the source of many of these ECM molecules. Histologically, abnormal extracellular matrix can be found between the RPE cells and the basement membrane (basal laminar deposits) and external to the basement membrane within the collagenous layers of the membrane (basal linear deposits) (Bressler et al., 1994; Green and Enger, 2005). Drusen therefore can be a localized accentuation of these deposits in AMD (Bressler et al., 1994).

The increase in thickness and change in composition of the Bruch's membrane in AMD can lead to a disruption of the exchange of molecules between choriocapillaris and the subretinal space (Starita et al., 1997).

In support of this model, it has been shown that the hydraulic conductivity of the Bruch's membrane falls exponentially with age. Measurements have shown that most of the resistance to water flow lies in the inner collagenous layer of the Bruch's membrane which is possibly due to accumulation of abnormal entrapped material within this plane (Starita et al., 1997). Therefore, the thickened Bruch's membrane in AMD may lead to a diffusion barrier, leading to RPE and retinal dysfunction (Pauleikhoff et al., 1999; Remulla et al., 1995).

#### **2.2.4 CNV formation**

Multiple factors have been proposed as promoters of new blood vessels formation in wet AMD. Changes in the ECM is one of the abnormalities seen in AMD which can lead to the formation of new blood vessels. The mechanism by which this phenomenon occurs is not completely understood but is likely to be a multifactorial. The risk of CNV in AMD increases with the increase in Drusen. Some drusen components and advanced glycation end products stimulate the production of angiogenic factors (Lu et al., 1998; Mousa et al., 1999). The increased thickness of Bruch's membrane can also lead to reductions in choriocapillary blood flow and hypoxia (Remulla et al., 1995). Hypoxia in turn can upregulate genes Ang-1 and Ang-2, with Ang-1 promoting maturation and stabilization of blood vessels, and Ang-2 conferring endothelial cell responsiveness to angiogenic factors (Hanahan, 1997; Maisonpierre et al.,1997). In addition, RPE cells are themselves known to produce angiogenic factors, such as VEGF, (Kim et al., 1999) which can lead to neovascularisation. High concentrations of VEGF and its receptors are found in CNV and RPE cells (Kliffen et al., 1997; Kvanta et al., 1996). Furthermore, anti-VEGF treatments prevent laser induced CNV formation in primate models of AMD (Krzystolik et al., 2002).

It has been shown that overexpression of VEGF in transgenic mice leads to the formation of aberrant choriocapillaries. However, these vessels are not capable of penetrating the intact Bruch's membrane (Schwesinger et al., 2001). Therefore, damage to Bruch's membrane due

IV collagen in the aging RPE basement membrane, particularly over the drusen (Pauleikhoff

There is an age dependent increase in type I collagen within the Bruch's membrane, with an increase in the thickness of the membrane from 2 micrometres at birth, to up to 6 micrometres in the elderly ages (Ramrattan et al., 1994). During aging , the membrane glycosaminglycans in Bruch's membrane increase in size, and there is an increase in the heparan sulphate proteoglycan content of the membrane (Hewitt et al., 1989). Furthermore, glycation end products can accumulate within the Bruch's membrane with aging, trapping

RPE cells themselves are the source of many of these ECM molecules. Histologically, abnormal extracellular matrix can be found between the RPE cells and the basement membrane (basal laminar deposits) and external to the basement membrane within the collagenous layers of the membrane (basal linear deposits) (Bressler et al., 1994; Green and Enger, 2005). Drusen therefore can be a localized accentuation of these deposits in AMD

The increase in thickness and change in composition of the Bruch's membrane in AMD can lead to a disruption of the exchange of molecules between choriocapillaris and the

In support of this model, it has been shown that the hydraulic conductivity of the Bruch's membrane falls exponentially with age. Measurements have shown that most of the resistance to water flow lies in the inner collagenous layer of the Bruch's membrane which is possibly due to accumulation of abnormal entrapped material within this plane (Starita et al., 1997). Therefore, the thickened Bruch's membrane in AMD may lead to a diffusion barrier, leading to RPE and retinal dysfunction (Pauleikhoff et al., 1999; Remulla et al., 1995).

Multiple factors have been proposed as promoters of new blood vessels formation in wet AMD. Changes in the ECM is one of the abnormalities seen in AMD which can lead to the formation of new blood vessels. The mechanism by which this phenomenon occurs is not completely understood but is likely to be a multifactorial. The risk of CNV in AMD increases with the increase in Drusen. Some drusen components and advanced glycation end products stimulate the production of angiogenic factors (Lu et al., 1998; Mousa et al., 1999). The increased thickness of Bruch's membrane can also lead to reductions in choriocapillary blood flow and hypoxia (Remulla et al., 1995). Hypoxia in turn can upregulate genes Ang-1 and Ang-2, with Ang-1 promoting maturation and stabilization of blood vessels, and Ang-2 conferring endothelial cell responsiveness to angiogenic factors (Hanahan, 1997; Maisonpierre et al.,1997). In addition, RPE cells are themselves known to produce angiogenic factors, such as VEGF, (Kim et al., 1999) which can lead to neovascularisation. High concentrations of VEGF and its receptors are found in CNV and RPE cells (Kliffen et al., 1997; Kvanta et al., 1996). Furthermore, anti-VEGF treatments prevent laser induced CNV formation in primate models of AMD (Krzystolik et al., 2002). It has been shown that overexpression of VEGF in transgenic mice leads to the formation of aberrant choriocapillaries. However, these vessels are not capable of penetrating the intact Bruch's membrane (Schwesinger et al., 2001). Therefore, damage to Bruch's membrane due

other macromolecules (King and Brownlee, 1996; Schmidt et al., 2000).

et al., 1999).

(Bressler et al., 1994).

**2.2.4 CNV formation** 

subretinal space (Starita et al., 1997).

to various factors in combination with the upregulation of VEGF, can synergistically lead to the choriocapillary CNVs penetrating the membrane and reaching the subretinal space (Schwesinger et al.,2001; Zarbin ,2004).

One of the molecules that has been studied extensively in our lab is a glycoprotein called tenascin C, known to be overexpressed in angiogenesis (Zagzag and Capo, 2002; Zagzaget al., 1996), neovascularisation and wound healing (Maseruka et al., 1997). Tenascin C deposition can occur in the Bruch's membrane in wet AMD on the basal side of RPE cells (Fasler-Kan et al., 2005) and in association with CNVs in the pathological Bruch's membrane (Nicolo et al., 2000). Tenascin C has been shown to prevent adhesion of RPE cells to extracellular matrix (Afshari et al 2010). Therefore accumulation of this molecule associated with CNV formation may play an important role in RPE loss from the Bruch's membrane seen in AMD (Afshari et al 2010).

In summary, different pathological processes during aging and in AMD can lead to modifications in the Bruch's membrane which ultimately becomes a less supportive environment for the RPE adhesion and function.

#### **3. Experimental models available for studying wet AMD**

#### **3.1** *In vitro* **and** *ex vivo* **models - Advantages vs disadvantages**

*In vitro* models have allowed development of simplified systems to study processes involved in wet AMD. Most *in vitro* models have focused on the role of angiogenesis and isolation of Bruch's membrane to assess adhesion and survival of RPE cells.

Tezel and Del priore first described methodology for accessing different layers of Bruch's membrane to allow *in vitro* assessment of RPE adhesion at different levels of Bruch's membrane. A combination of enzymatic treatment and mechanical techniques were used to expose each layer sequentially starting from the top basal lamina and moving to deeper structures. Using this technique, it was shown that deeper layers of Bruch's membrane are less supportive of RPE attachment (Del priore et al 1998; Tezel TH 1999 FEB; Tezel TH 1999 March). RPE cell adhesion to Bruch's membrane may play a detrimental role both in AMD and following RPE transplantation.

An alternative way of accessing Bruch's membrane used in our lab is the water lysis technique (Afshari et al 2010). In this method, eye globes are dissected out and separated from their muscle attachments. The anterior chamber is then dissected away leaving the posterior chamber and retina and Bruch's-choroid-sclera. Retinal layer is then carefully removed leaving the Bruch's-choroid-sclera trilaminar structure which can be subsequently exposed to water. Exposure to water leads to lysis of endogenous RPE cells. Lysed RPE cells are then flushed away from the surface of Bruch's membrane using a mini water jet. This procedure therefore results in formation of a denuded Bruch's membrane which can allow further experiments such as transplanting exogenous RPE cells to assess adhesion and migration of the transplanted cells (Afshari et al 2010). The advantages of this technique is that minimal treatment of the tissue is required with preservation of natural Bruch's membrane. In addition the preparation of the Bruch's membrane for adhesion and migration assay is a short procedure. Immunostaining of both frozen sections and electron microscopy of the membranes following water treatment have confirmed complete removal

Wet Age Related Macular Degeneration 13

Introduction of this mitogen can lead to CNV formation from choriocapillaries (Tanaka N 2006). By generating transgenic mice expressing Hpk1 in retina, Tanaka et al were able to show that Hpk1 promotes development of CNV with no effect on retinal vasculature. Interestingly, these mice also show increased levels of lipofuscin which is also seen in AMD

One of the most interesting examples of transgenic mice used in studying wet AMD is the ccr2/ccl2 transgenic mice which are unable to recruit macrophages to RPE layer and Bruch's membrane. This leads to accumulation of C5a and Immunoglobulin G which in turn leads to

An alternative method of CNV formation is application of laser to generate a focal area of burn within the Bruch's membrane which in turn leads to CNV formation. This technique over the years has become one of the most standard and widely used techniques in studying wet AMD. Various laser treatments using krypton, argon and diode have all been able to induce CNV formation in mice, rats, pigs and monkeys (Dobi et al 1989; Frank et al 1989;Ryan et al. 1979; Saishin et al 2003). To initiate CNV formation using laser, it is necessary for RPE layer, Bruch's membrane and the underlying choroid to be damaged by the laser to allow penetration and initiation of new blood vessel formation. The laser induced CNV formation is VEGF mediated, as different methods of blocking VEGF using peptides and antibodies in mice, rats and monkeys are all able to block the

Since defects in Bruch's membrane in age related macular degeneration leads to RPE loss, replacement of RPE cells by transplantation has been proposed as a technique to prevent secondary photoreceptor death. In the past two decades, studies in various animal models of retinal degeneration and RPE loss have shown that RPE cell replacement may be a feasible technique to prevent a secondary photoreceptor loss due to RPE damage (Lund et

Li et al in 1988 demonstrated that RPE transplantation in young neonatal and adult rats allows a repopulation of denuded areas on the Bruch's membrane and prevent the photoreceptor degeneration in dystrophic RCS rat models of AMD (Liand Turner, 1988a, b). In separate studies, Castillo et al have shown that transplantation of adult young human RPE cells derived from cadaveric eye samples, into the dystrophic RCS rats can salvage the

Furthermore, subretinal transplantation of the RPE cell line ARPE-19, the most widely used adult human RPE cell line, in dystrophic RCS rats can rescue the photoreceptors (Wang et al., 2005). Other animal models, such as rabbit models of RPE damage, showed that mechanical debridement of the Bruch's membrane followed by autologous RPE transplantation leads to the repopulation of debrided Bruch's membrane with preservation

In humans patients with AMD, the formation of choroidal new vessels is part of the pathology of advanced wet AMD. The removal of CNVs has also been carried out in human

stimulation of VEGF production (Ambati 2003; Takeda et al. 2009).

neovascularisation process (Hua J 2010; Goody RJ 2011).

**4.1 Surgical and cellular transplantation/replacement** 

photoreceptor loss in this model (Castillo et al., 1997).

of photoreceptors (Phillips et al., 2003).

**4. Treatments available for AMD and their mode of action** 

(Tanaka N 2006).

al., 2001).

of endogenous RPE layer therefore creating a suitable environment for transplanting exogenous cells (Afshari et al 2010). However for assessment of adhesion on different layers such as deeper collagen layers of Bruch's membrane, methodology by Tezel and Del priore et al can be used (Del priore et al 1998; Tezel TH 1999 FEB; Tezel TH 1999 March).

Although much has been learned from the use of eyes derived from experimental animals such as rats and rabbits, a major problem faced is the unique human age related changes and AMD related pathological processes that have been hard to recapitulate in animal models. Therefore recent attention has been on use of human derived Bruch's membrane and *ex vivo* models whereby pathological or normal samples can be used from donors. A great advantage of this technique is that good methodology exists for isolation of layers of Bruch's membrane, and eyes from various stages of the disease can be studied. A disadvantage of using human samples is the difficulty in obtaining high quality tissue before post mortem deterioration occurs.

#### **3.2** *In vivo* **models - Advantages vs disadvantages**

*In vivo* animal models have been used widely in studying AMD. Creating animal models specific for AMD has been a difficult task to achieve. One of the older animal models used in AMD research is Royal College of Surgeons rats (RCS rats) where RPE cells are gradually lost over time along with photoreceptors. RCS rats have been used in RPE transplantation experiments widely to assess efficiency of transplanted cells in replacing the lost endogenous RPE cells and preventing photoreceptor loss (Li and Turner 1988). However these rats are a better model for studying retinitis pigmentosa and therefore may differ considerably with regards to pathology from AMD.

Another used animal model comprises of mechanically scratching the RPE layer. This allows creation of focal areas devoid of RPE cells allowing studying various transplantation or pharmacological treatments. Rabbits are used generally in this model (Philips 2003) due to bigger size of the eye globes allowing easier access.

None of the models above recapitulate the neovascularisation seen in wet AMD. However recently more models have emerged which reproduce the neovascularisation process. Some of these models use growth factors such as b-fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) to induce the endothelial cells proliferation and migration to promote CNV formation in rats, rabbits and monkeys (Montezumas.R 2009, Edwards A. 2007, Lassota N 2008, Baba T 2010). Over the years different techniques have been used to deliver growth factors ranging from direct injections, lentiviral vectors, cells secreting growth factors or transgenic animals secreting the VEGF (Spilsbury 2000; julien 2008; Okamoto et al1997; Cui et al 2000) .

Newer techniques which can stimulate CNV formation include injection of matrigel subretinally which allows a suitable environment for blood vessels to grow into (Cao J 2010). An alternative to this has been use of polyethylene glycol injections subretinally which leads to activation of complement cascade and generation of VEGF leading to CNV formation in mouse (Lyzogubov et al 2011).

Multiple transgenic mice lines also have been created which produce CNV through different methods. One of such animal models is use of transgenic mice producing mitogen prokineticin 1 (Hpk1) which specifically stimulates fenestrated endothelial cells.

of endogenous RPE layer therefore creating a suitable environment for transplanting exogenous cells (Afshari et al 2010). However for assessment of adhesion on different layers such as deeper collagen layers of Bruch's membrane, methodology by Tezel and Del priore

Although much has been learned from the use of eyes derived from experimental animals such as rats and rabbits, a major problem faced is the unique human age related changes and AMD related pathological processes that have been hard to recapitulate in animal models. Therefore recent attention has been on use of human derived Bruch's membrane and *ex vivo* models whereby pathological or normal samples can be used from donors. A great advantage of this technique is that good methodology exists for isolation of layers of Bruch's membrane, and eyes from various stages of the disease can be studied. A disadvantage of using human samples is the difficulty in obtaining high quality tissue

*In vivo* animal models have been used widely in studying AMD. Creating animal models specific for AMD has been a difficult task to achieve. One of the older animal models used in AMD research is Royal College of Surgeons rats (RCS rats) where RPE cells are gradually lost over time along with photoreceptors. RCS rats have been used in RPE transplantation experiments widely to assess efficiency of transplanted cells in replacing the lost endogenous RPE cells and preventing photoreceptor loss (Li and Turner 1988). However these rats are a better model for studying retinitis pigmentosa and therefore may differ

Another used animal model comprises of mechanically scratching the RPE layer. This allows creation of focal areas devoid of RPE cells allowing studying various transplantation or pharmacological treatments. Rabbits are used generally in this model (Philips 2003) due to

None of the models above recapitulate the neovascularisation seen in wet AMD. However recently more models have emerged which reproduce the neovascularisation process. Some of these models use growth factors such as b-fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) to induce the endothelial cells proliferation and migration to promote CNV formation in rats, rabbits and monkeys (Montezumas.R 2009, Edwards A. 2007, Lassota N 2008, Baba T 2010). Over the years different techniques have been used to deliver growth factors ranging from direct injections, lentiviral vectors, cells secreting growth factors or transgenic animals secreting the VEGF (Spilsbury 2000; julien 2008;

Newer techniques which can stimulate CNV formation include injection of matrigel subretinally which allows a suitable environment for blood vessels to grow into (Cao J 2010). An alternative to this has been use of polyethylene glycol injections subretinally which leads to activation of complement cascade and generation of VEGF leading to CNV formation in

Multiple transgenic mice lines also have been created which produce CNV through different methods. One of such animal models is use of transgenic mice producing mitogen prokineticin 1 (Hpk1) which specifically stimulates fenestrated endothelial cells.

et al can be used (Del priore et al 1998; Tezel TH 1999 FEB; Tezel TH 1999 March).

before post mortem deterioration occurs.

**3.2** *In vivo* **models - Advantages vs disadvantages** 

considerably with regards to pathology from AMD.

bigger size of the eye globes allowing easier access.

Okamoto et al1997; Cui et al 2000) .

mouse (Lyzogubov et al 2011).

Introduction of this mitogen can lead to CNV formation from choriocapillaries (Tanaka N 2006). By generating transgenic mice expressing Hpk1 in retina, Tanaka et al were able to show that Hpk1 promotes development of CNV with no effect on retinal vasculature. Interestingly, these mice also show increased levels of lipofuscin which is also seen in AMD (Tanaka N 2006).

One of the most interesting examples of transgenic mice used in studying wet AMD is the ccr2/ccl2 transgenic mice which are unable to recruit macrophages to RPE layer and Bruch's membrane. This leads to accumulation of C5a and Immunoglobulin G which in turn leads to stimulation of VEGF production (Ambati 2003; Takeda et al. 2009).

An alternative method of CNV formation is application of laser to generate a focal area of burn within the Bruch's membrane which in turn leads to CNV formation. This technique over the years has become one of the most standard and widely used techniques in studying wet AMD. Various laser treatments using krypton, argon and diode have all been able to induce CNV formation in mice, rats, pigs and monkeys (Dobi et al 1989; Frank et al 1989;Ryan et al. 1979; Saishin et al 2003). To initiate CNV formation using laser, it is necessary for RPE layer, Bruch's membrane and the underlying choroid to be damaged by the laser to allow penetration and initiation of new blood vessel formation. The laser induced CNV formation is VEGF mediated, as different methods of blocking VEGF using peptides and antibodies in mice, rats and monkeys are all able to block the neovascularisation process (Hua J 2010; Goody RJ 2011).

#### **4. Treatments available for AMD and their mode of action**

#### **4.1 Surgical and cellular transplantation/replacement**

Since defects in Bruch's membrane in age related macular degeneration leads to RPE loss, replacement of RPE cells by transplantation has been proposed as a technique to prevent secondary photoreceptor death. In the past two decades, studies in various animal models of retinal degeneration and RPE loss have shown that RPE cell replacement may be a feasible technique to prevent a secondary photoreceptor loss due to RPE damage (Lund et al., 2001).

Li et al in 1988 demonstrated that RPE transplantation in young neonatal and adult rats allows a repopulation of denuded areas on the Bruch's membrane and prevent the photoreceptor degeneration in dystrophic RCS rat models of AMD (Liand Turner, 1988a, b). In separate studies, Castillo et al have shown that transplantation of adult young human RPE cells derived from cadaveric eye samples, into the dystrophic RCS rats can salvage the photoreceptor loss in this model (Castillo et al., 1997).

Furthermore, subretinal transplantation of the RPE cell line ARPE-19, the most widely used adult human RPE cell line, in dystrophic RCS rats can rescue the photoreceptors (Wang et al., 2005). Other animal models, such as rabbit models of RPE damage, showed that mechanical debridement of the Bruch's membrane followed by autologous RPE transplantation leads to the repopulation of debrided Bruch's membrane with preservation of photoreceptors (Phillips et al., 2003).

In humans patients with AMD, the formation of choroidal new vessels is part of the pathology of advanced wet AMD. The removal of CNVs has also been carried out in human

Wet Age Related Macular Degeneration 15

reduced when RPE cells are seeded on aged membranes than the young membranes (Sun, et

These functional differences are further backed up by the changes in gene expression between RPE cells cultured on aged and young membranes. It has been shown that the RPE cells seeded on aged membranes up-regulate 12 genes and downregulate 8 genes compared to RPE cells cultured on membranes derived from young donors suggesting the differences

Therefore, it is evident that there is a significant age-dependent decline in the Bruch's membrane's ability to support the RPE cell adhesion and function, and therefore RPE loss and dysfunction in AMD can be at least partially reflective of changes within the membrane. These changes in Bruch's membrane therefore pose an obstacle for the transplanted RPE

In addition, data from our lab and others have shown that in wet AMD, there is increased deposition of a glycoprotein associated with neovascularisation. This glycoprotein named tenascin C is deposited on the upper layer of Bruch's membrane. Using purified tenascin C, we were able to show that human RPE cells lack the necessary integrins to attach to surfaces coated with this glycoprotein and therefore deposition of this molecule in pathological AMD Bruch's membrane further reduces the chance of adhesion. Using *in vitro* assays we were able to show that if RPE cells are engineered to express a necessary receptor called alpha9beta1 integrin for tenascin C, they are able to attach following transplantation to the wet AMD derived Bruch's membrane where as in the absence of this receptor, control RPE

In addition to changes mentioned above, surgical techniques used in removal of CNVs have been shown to damage the normal architecture of Bruch's membrane. It is well established that surgical removal of CNVs in the wet AMD generally leads to excision of the basement membrane of the Bruch's membrane (Grossniklaus et al., 1994). Tsukahara et al using *ex vivo* models of aged Bruch's membrane have shown that the resurfacing of the Bruch's membrane is highly dependent on whether the basement membrane is intact or removed. The adhesion of RPE cells was much higher on aged Bruch's membrane if the basement membrane was not damaged and removed (Tsukahara et al., 2002). Therefore, one of the limitations of the CNV removal procedure is the iatrogenic removal of the laminin rich basement membrane, which reduces the chance of adhesion of RPE cells transplanted

In addition to the removal of the laminin rich basement membrane of Bruch'smembrane, the surgical procedures also lead to the exposure of deeper layers of the Bruch's membrane. Various studies have assessed the adhesion rate and the survival of RPE cells on different layers of the Bruch's membrane. They have revealed that RPE cell reattachment is the highest on the uppermost layers of the Bruch's membrane which include basement membrane. As deeper layers are exposed, this adhesion rate decreases (Del Priore and Tezel, 1998). Thus, following CNV removal, depending on which layer of the Bruch's membrane is exposed , the outcome of adhesion will differ which diminishes the chances of fast and efficient adhesion of the RPE cells following transplantation (Del Priore and Tezel, 1998).

RPE cells are known to attach to the human Bruch's membrane through beta1 integrinmediated interaction, with extracellular ligands such as laminin, fibronectin, vitronectin and

cells, which require fast attachment and adhesion, to survive post-transplantation.

cells were unable to attach to the membrane effectively (Afshari et al 2010).

subsequently into the subretinal space.

between ages are also reflected at gene level (Cai and Del Priore, 2006).

al., 2007).

patients with AMD. This can be followed by autologous transplantation of RPE cells, either harvested from the periphery of the Bruch's membrane which is not affected by the disease process (Binder et al., 2007), or from RPE cells from other donors (Algvere et al.,1994).

Algevere et al at in 1994 assessed the effect of human fetal RPE transplantation in 5 patients with AMD after the removal of CNVs. Human fetal RPE cells survived up to 3 months and covered the denuded areas of the Bruch's membrane (Algvere et al., 1994).

Other studies have also assessed the effect of adult autologous transplantation of RPE cells in AMD. It has been shown that autolgous transplantation following the removal of CNVs is a feasible technique and associated with some visual acuity improvement (Binder et al., 2004).

In 2007 Maclaren et al carried out autologous transplantation of the RPE cells, following submacular CNV excision, and reported viable grafts at 6 months time point and some level of visual function improvement in some patients. However, the complications associated with the surgery remained high (MacLaren et al., 2007).

RPE transplantation has traditionally been carried out as cell suspension but, due to problems with RPE attachment to Bruch's membrane, more recently RPE-choroid sheets have been tried as a means of delivering RPE cells (Treumer et al 2007). In 2011, Falkner-Radler et al, carried out a study comparing RPE cell suspension with that of RPE-choroid sheet transplantation. This study showed that anatomical and functional outcome in both cases were comparable with no significant difference between the two techniques in humans (Falkner-RadlerCl 2011).

Despite some improvements gained in the visual function, the results from the CNV removal combined with RPE transplantation, have not been as successful as those observed with animal models. This may be due to age related changes specific to human AMD which are absent in the animal models used in studying AMD and RPE transplantation.

RPE transplantation as a therapeutic technique faces major limitations, including poor adhesion of RPE cells when transplanted subretinally. Studies have shown that RPE cells require rapid adhesion to avoid apoptosis (Tezel and Del Priore, 1997,1999). Therefore, there is a limited time period after subretinal injection during which RPE cells need to reattach before undergoing cell death.

The lack of adhesion following transplantation is likely to be multifactorial due to the molecular changes resulting from pathological age related changes in the membrane, and other changes contributed by the disturbance of normal architecture of the membrane from the surgery.

Various studies using *ex vivo* models have demonstrated major differences between RPE and Bruch's membrane in patients from different ages, emphasizing the important role of aging in the pathological process. Studies by Gullapalli et al have shown that aged submacular human Bruch's membrane does not support adhesion, survival and differentiation of fetal RPE cells effectively (Gullapalli et al., 2005). Multiple studies have shown that RPE cell adhesion to the Bruch's membrane is reduced on aged membranes, when compared to the membrane derived from younger donors (Del Priore and Tezel,1998; Tezel et al., 1999).

In addition to changes in adhesion, survival and differentiation, it has been shown that the capacity of RPE cells to phagocytose the shed outer segment of rod photoreceptors is

patients with AMD. This can be followed by autologous transplantation of RPE cells, either harvested from the periphery of the Bruch's membrane which is not affected by the disease process (Binder et al., 2007), or from RPE cells from other donors (Algvere et al.,1994).

Algevere et al at in 1994 assessed the effect of human fetal RPE transplantation in 5 patients with AMD after the removal of CNVs. Human fetal RPE cells survived up to 3 months and

Other studies have also assessed the effect of adult autologous transplantation of RPE cells in AMD. It has been shown that autolgous transplantation following the removal of CNVs is a feasible technique and associated with some visual acuity improvement (Binder et al.,

In 2007 Maclaren et al carried out autologous transplantation of the RPE cells, following submacular CNV excision, and reported viable grafts at 6 months time point and some level of visual function improvement in some patients. However, the complications associated

RPE transplantation has traditionally been carried out as cell suspension but, due to problems with RPE attachment to Bruch's membrane, more recently RPE-choroid sheets have been tried as a means of delivering RPE cells (Treumer et al 2007). In 2011, Falkner-Radler et al, carried out a study comparing RPE cell suspension with that of RPE-choroid sheet transplantation. This study showed that anatomical and functional outcome in both cases were comparable with no significant difference between the two techniques in humans

Despite some improvements gained in the visual function, the results from the CNV removal combined with RPE transplantation, have not been as successful as those observed with animal models. This may be due to age related changes specific to human AMD which

RPE transplantation as a therapeutic technique faces major limitations, including poor adhesion of RPE cells when transplanted subretinally. Studies have shown that RPE cells require rapid adhesion to avoid apoptosis (Tezel and Del Priore, 1997,1999). Therefore, there is a limited time period after subretinal injection during which RPE cells need to reattach

The lack of adhesion following transplantation is likely to be multifactorial due to the molecular changes resulting from pathological age related changes in the membrane, and other changes contributed by the disturbance of normal architecture of the membrane from

Various studies using *ex vivo* models have demonstrated major differences between RPE and Bruch's membrane in patients from different ages, emphasizing the important role of aging in the pathological process. Studies by Gullapalli et al have shown that aged submacular human Bruch's membrane does not support adhesion, survival and differentiation of fetal RPE cells effectively (Gullapalli et al., 2005). Multiple studies have shown that RPE cell adhesion to the Bruch's membrane is reduced on aged membranes, when compared to the membrane derived from younger donors (Del Priore and Tezel,1998; Tezel et al., 1999).

In addition to changes in adhesion, survival and differentiation, it has been shown that the capacity of RPE cells to phagocytose the shed outer segment of rod photoreceptors is

are absent in the animal models used in studying AMD and RPE transplantation.

covered the denuded areas of the Bruch's membrane (Algvere et al., 1994).

with the surgery remained high (MacLaren et al., 2007).

2004).

(Falkner-RadlerCl 2011).

before undergoing cell death.

the surgery.

reduced when RPE cells are seeded on aged membranes than the young membranes (Sun, et al., 2007).

These functional differences are further backed up by the changes in gene expression between RPE cells cultured on aged and young membranes. It has been shown that the RPE cells seeded on aged membranes up-regulate 12 genes and downregulate 8 genes compared to RPE cells cultured on membranes derived from young donors suggesting the differences between ages are also reflected at gene level (Cai and Del Priore, 2006).

Therefore, it is evident that there is a significant age-dependent decline in the Bruch's membrane's ability to support the RPE cell adhesion and function, and therefore RPE loss and dysfunction in AMD can be at least partially reflective of changes within the membrane. These changes in Bruch's membrane therefore pose an obstacle for the transplanted RPE cells, which require fast attachment and adhesion, to survive post-transplantation.

In addition, data from our lab and others have shown that in wet AMD, there is increased deposition of a glycoprotein associated with neovascularisation. This glycoprotein named tenascin C is deposited on the upper layer of Bruch's membrane. Using purified tenascin C, we were able to show that human RPE cells lack the necessary integrins to attach to surfaces coated with this glycoprotein and therefore deposition of this molecule in pathological AMD Bruch's membrane further reduces the chance of adhesion. Using *in vitro* assays we were able to show that if RPE cells are engineered to express a necessary receptor called alpha9beta1 integrin for tenascin C, they are able to attach following transplantation to the wet AMD derived Bruch's membrane where as in the absence of this receptor, control RPE cells were unable to attach to the membrane effectively (Afshari et al 2010).

In addition to changes mentioned above, surgical techniques used in removal of CNVs have been shown to damage the normal architecture of Bruch's membrane. It is well established that surgical removal of CNVs in the wet AMD generally leads to excision of the basement membrane of the Bruch's membrane (Grossniklaus et al., 1994). Tsukahara et al using *ex vivo* models of aged Bruch's membrane have shown that the resurfacing of the Bruch's membrane is highly dependent on whether the basement membrane is intact or removed. The adhesion of RPE cells was much higher on aged Bruch's membrane if the basement membrane was not damaged and removed (Tsukahara et al., 2002). Therefore, one of the limitations of the CNV removal procedure is the iatrogenic removal of the laminin rich basement membrane, which reduces the chance of adhesion of RPE cells transplanted subsequently into the subretinal space.

In addition to the removal of the laminin rich basement membrane of Bruch'smembrane, the surgical procedures also lead to the exposure of deeper layers of the Bruch's membrane. Various studies have assessed the adhesion rate and the survival of RPE cells on different layers of the Bruch's membrane. They have revealed that RPE cell reattachment is the highest on the uppermost layers of the Bruch's membrane which include basement membrane. As deeper layers are exposed, this adhesion rate decreases (Del Priore and Tezel, 1998). Thus, following CNV removal, depending on which layer of the Bruch's membrane is exposed , the outcome of adhesion will differ which diminishes the chances of fast and efficient adhesion of the RPE cells following transplantation (Del Priore and Tezel, 1998).

RPE cells are known to attach to the human Bruch's membrane through beta1 integrinmediated interaction, with extracellular ligands such as laminin, fibronectin, vitronectin and

Wet Age Related Macular Degeneration 17

trials comparing these two drugs directly together. Bevacizumab, which also blocks VEGF and is considerably cheaper than its counterparts, has also been used off licence for treating wet AMD although originally it was licensed for colorectal carcinoma (Avery 2006, Emerson 2007). Although multiple studies have shown efficacy of this monoclonal antibody in reducing neovascularisation, the safety profile of this antibody is not as clear as other two

With increasing aging population, the number of patients with AMD is likely to rise sharply. The projected number of advanced AMD cases is likely to rise by 50% by year 2020 (Friedman et al 2004). Therefore with increasing incidence of this condition, screening programs may be of value to allow early detection and treatment of this condition. This is of paramount importance as early detection has been shown to be associated with a better

With recent advances in cell transplantation and knowledge of stem cells, it may be possible that stem cell derived RPE cells can be used in the treatment of AMD (Lee and Maclaren 2011). Use of these cells may be of benefit as they have the potential to replace the lost cells and may not be hindered by the obstacles such as poor adhesion faced with cadaveric or donor derived RPE cells. For dry AMD, cell transplantation strategies are also undergoing clinical trials in several centres worldwide . Strategies to compare improve the survival and adhesion of transplanted cells to damaged Bruch's membrane are a key focus of our ongoing

Manipulation of integrins on RPE cells or genetic engineering of transplanted cells is a new field that holds promise in overcoming the obstacles faced in cell transplantation. Activating integrins by enhancing their function or introduction of new subunits of integrins into RPE cells have been shown to overcome the poor attachment and integration of RPE cells over Bruch's membrane (Afshari et al 2010; Fang et al 2009). It is therefore possible that with better understanding of RPE biology, adhesion and survival of cells following

With the advent of the new therapies such as monoclonal anti-VEGF treatments, major advances have occurred in the treatment of wet AMD. At this point the challenges reside in wide access and affordable costs to allow early recognition and prevention of loss of vision at an early stage. Currently repeated injections of monoclonal antibodies limit their use in areas where access to such therapies is limited. With better understanding and experience of using such therapies, it is hoped that treatments with longer half lives and more affordable

Afshari FT, Kwok JC, Andrews MR, Blits B, Martin KR, Faissner A, Ffrench-Constant C,

Fawcett JW. Integrin activation or alpha 9 expression allows retinal pigmented epithelial cell adhesion on Bruch's membrane in wet age-related macular

(Mitchell P 2011).

work.

**5. Problems and challenges for future** 

outcome and prognosis (Wong et al 2008).

transplantation could be improved.

**6. References** 

prices can be available to increasing aging population.

degeneration. Brain. 2010 Feb;133(Pt 2):448-64.

collagen IV (Ho and DelPriore, 1997). Tezel et al have demonstrated that laminin and fibronectin supported the adhesion of RPE cells best and prevented cellular apoptosis (Tezel and Del Priore,1997). Since the upper most layers of the Bruch's membrane are rich in laminin and fibronectin, removal of basement membrane combined with the exposure of deeper less adhesive substrates, limits adhesion following transplantation.

Therefore, there is a great need for promoting cell adhesion post transplantation to allow resurfacing and seeding of the pathologically and surgically altered membranes. Multiple problems faced with transplantation therefore haves lead to more attention on pharmacological and less invasive techniques to halt the CNV formation.

#### **4.2 Photodynamic therapy and laser treatment**

Laser photocoagulation is one of the techniques that was developed to treated neovascularisation problem in wet AMD. Since this technique leads to full thickness retinal burns, this can lead to loss of visual acuity if carried out in foveal region and therefore it is reserved for extrafoveal CNVs. In addition, there is a high rate of recurrence of CNVs following treatment with this method (Vedula SS and Krzystolik M 2011). However this technique is effective in reducing the progression of non-subfoveal CNVs compared to observation alone (Virgil 2007; Verdula SS and Krzystolik M 2011).

Photodynamic therapy on the other hand is a technique that works by injecting a photosensitive dye intravenously which preferentially binds to CNVs. On exposure of the eye to laser light, the dye can be activated leading to obliteration of the CNVs. This technique has the advantage of causing minimal trauma to normal choroid and membrane and the overlying retina. It therefore can be used for subfoveal lesions. The disadvantage with this technique is the necessity to repeat this procedure at least multiple times due to high rate of recurrence (TAP 1999;Verdula SS 2011).

#### **4.3 Anti-VEGF monoclonal antibodies**

One of the most recent approaches in battling wet AMD is the use of anti-VEGF monoclonal antibodies. Vascular endothelial growth factor has been shown to be involved in promoting formation of new blood vessels. The source of VEGF in AMD is believed to be the RPE cells themselves. Multiple studies have demonstrated presence of VEGF in RPE cells and its association with CNVs (Kim et al. 1999; Klifen 1997; Kvanata 1996). Although VEGF is necessary for neovascularisation, animal research shows that in the presence of intact normal Bruch's membrane, blood vessels will not invade the subretinal area and therefore a pathological process must render the membrane permeable to invading growing new blood vessels in AMD setting (Schwesinger 2001). Regardless of this finding, use of blocking agents against VEGF or its receptor holds promise in halting neovascularisation.

Animal studies have shown that blocking VEGF using different approaches can halt the neovascularisation process. Multiple clinical trials have assessed efficacy and safety of anti-VEGF monoclonal antibodies which include Bevacizumab, ranibizumab, pegabtanib (Vendula SS and KrzystolikM 2011). A recent systematic review of randomised controlled trials compared recent trials using anti-VEGF in wet AMD. Pegabtanib and Ranibizumab were shown to be both effective in reducing the neovascularisation with improvements in visual acuity and quality of life (Vendula SS and Krzystolik M 2011). There are currently no

collagen IV (Ho and DelPriore, 1997). Tezel et al have demonstrated that laminin and fibronectin supported the adhesion of RPE cells best and prevented cellular apoptosis (Tezel and Del Priore,1997). Since the upper most layers of the Bruch's membrane are rich in laminin and fibronectin, removal of basement membrane combined with the exposure of

Therefore, there is a great need for promoting cell adhesion post transplantation to allow resurfacing and seeding of the pathologically and surgically altered membranes. Multiple problems faced with transplantation therefore haves lead to more attention on

Laser photocoagulation is one of the techniques that was developed to treated neovascularisation problem in wet AMD. Since this technique leads to full thickness retinal burns, this can lead to loss of visual acuity if carried out in foveal region and therefore it is reserved for extrafoveal CNVs. In addition, there is a high rate of recurrence of CNVs following treatment with this method (Vedula SS and Krzystolik M 2011). However this technique is effective in reducing the progression of non-subfoveal CNVs compared to

Photodynamic therapy on the other hand is a technique that works by injecting a photosensitive dye intravenously which preferentially binds to CNVs. On exposure of the eye to laser light, the dye can be activated leading to obliteration of the CNVs. This technique has the advantage of causing minimal trauma to normal choroid and membrane and the overlying retina. It therefore can be used for subfoveal lesions. The disadvantage with this technique is the necessity to repeat this procedure at least multiple times due to

One of the most recent approaches in battling wet AMD is the use of anti-VEGF monoclonal antibodies. Vascular endothelial growth factor has been shown to be involved in promoting formation of new blood vessels. The source of VEGF in AMD is believed to be the RPE cells themselves. Multiple studies have demonstrated presence of VEGF in RPE cells and its association with CNVs (Kim et al. 1999; Klifen 1997; Kvanata 1996). Although VEGF is necessary for neovascularisation, animal research shows that in the presence of intact normal Bruch's membrane, blood vessels will not invade the subretinal area and therefore a pathological process must render the membrane permeable to invading growing new blood vessels in AMD setting (Schwesinger 2001). Regardless of this finding, use of blocking

Animal studies have shown that blocking VEGF using different approaches can halt the neovascularisation process. Multiple clinical trials have assessed efficacy and safety of anti-VEGF monoclonal antibodies which include Bevacizumab, ranibizumab, pegabtanib (Vendula SS and KrzystolikM 2011). A recent systematic review of randomised controlled trials compared recent trials using anti-VEGF in wet AMD. Pegabtanib and Ranibizumab were shown to be both effective in reducing the neovascularisation with improvements in visual acuity and quality of life (Vendula SS and Krzystolik M 2011). There are currently no

agents against VEGF or its receptor holds promise in halting neovascularisation.

deeper less adhesive substrates, limits adhesion following transplantation.

pharmacological and less invasive techniques to halt the CNV formation.

observation alone (Virgil 2007; Verdula SS and Krzystolik M 2011).

**4.2 Photodynamic therapy and laser treatment** 

high rate of recurrence (TAP 1999;Verdula SS 2011).

**4.3 Anti-VEGF monoclonal antibodies** 

trials comparing these two drugs directly together. Bevacizumab, which also blocks VEGF and is considerably cheaper than its counterparts, has also been used off licence for treating wet AMD although originally it was licensed for colorectal carcinoma (Avery 2006, Emerson 2007). Although multiple studies have shown efficacy of this monoclonal antibody in reducing neovascularisation, the safety profile of this antibody is not as clear as other two (Mitchell P 2011).

#### **5. Problems and challenges for future**

With increasing aging population, the number of patients with AMD is likely to rise sharply. The projected number of advanced AMD cases is likely to rise by 50% by year 2020 (Friedman et al 2004). Therefore with increasing incidence of this condition, screening programs may be of value to allow early detection and treatment of this condition. This is of paramount importance as early detection has been shown to be associated with a better outcome and prognosis (Wong et al 2008).

With recent advances in cell transplantation and knowledge of stem cells, it may be possible that stem cell derived RPE cells can be used in the treatment of AMD (Lee and Maclaren 2011). Use of these cells may be of benefit as they have the potential to replace the lost cells and may not be hindered by the obstacles such as poor adhesion faced with cadaveric or donor derived RPE cells. For dry AMD, cell transplantation strategies are also undergoing clinical trials in several centres worldwide . Strategies to compare improve the survival and adhesion of transplanted cells to damaged Bruch's membrane are a key focus of our ongoing work.

Manipulation of integrins on RPE cells or genetic engineering of transplanted cells is a new field that holds promise in overcoming the obstacles faced in cell transplantation. Activating integrins by enhancing their function or introduction of new subunits of integrins into RPE cells have been shown to overcome the poor attachment and integration of RPE cells over Bruch's membrane (Afshari et al 2010; Fang et al 2009). It is therefore possible that with better understanding of RPE biology, adhesion and survival of cells following transplantation could be improved.

With the advent of the new therapies such as monoclonal anti-VEGF treatments, major advances have occurred in the treatment of wet AMD. At this point the challenges reside in wide access and affordable costs to allow early recognition and prevention of loss of vision at an early stage. Currently repeated injections of monoclonal antibodies limit their use in areas where access to such therapies is limited. With better understanding and experience of using such therapies, it is hoped that treatments with longer half lives and more affordable prices can be available to increasing aging population.

#### **6. References**

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Wet Age Related Macular Degeneration 19

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**2** 

Suofu Qin

*USA* 

**Pathogenic Roles of Sterile Inflammation in** 

**Etiology of Age-Related Macular Degeneration** 

*Retinal Disease Research, Department of Biological Sciences, Allergan, Inc., Irvine, CA* 

Inflammation is vital for host defense against invasive pathogens via the recruitment of innate inflammatory cells, which in turn phagocytose infectious agents and produce additional cytokines that then activate adaptive immune responses. Inflammation is also crucial to protect cells and facilitate wound healing as a result of mechanic or chemical injury. Because of the absence of infection, the inflammation induced by metabolic or chemical injury has been termed as 'sterile inflammation' to distinguish from that induced by pathogens. Similar to microbially induced inflammation, the immune system has evolved mechanisms to sense necrotic cell death by responding with innate and adaptive immune response, which is marked by the recruitment of macrophages and the production of inflammatory cytokines. However, unresolved and persistent inflammation due to un-removal or un-containment of the offending agents would turn it into a destructive process that is detrimental to the host. The production of reactive oxygen species (ROS), proteases and inflammatory cytokines causes tissue destruction and fibrosis. Thus, sterile inflammation has been demonstrated to be associated with human diseases such as cardiac ischaemia–reperfusion injury, the restoration of blood flow causes tissue destruction as a result of enhanced production of ROS and

Sterile inflammation in the etiology of age-related macular degeneration (AMD) has been highlighted by the observations that individuals with genetic mutation in complement factor H confer a significantly higher risky for AMD (Montezuma et al., 2007), an idiopathic retinal degenerative disease that leads to irreversible, profound vision loss in people over 60 year old in developed countries (Evans & Wormald, 1996). AMD occurs in two major forms: atrophic (dry) AMD and exudative (wet) AMD. The atrophic AMD is characterized by RPE atrophy and subjacent photoreceptor degeneration and accounts for approximately 25% of cases with severe central vision loss (Klein et al., 1997). Exudative AMD, which accounts for approximately 75% of cases with severe central vision loss (Klein et al., 1997), is characterized by choroidal neovascularization (CNV) and retinal hemorrhage. These two forms of AMD are both part of the same disease process and share similar risk factors for their development. Although the vision loss results from photoreceptor damage in the central retina, the initial pathogenesis of AMD has been proposed to involve the degeneration of retinal pigment epithelial (RPE) cells (Hageman et al., 2001). The RPE cells *in vivo* has limited regenerating capability upon damage as they are in general post-mitotic and their mitochondria are very susceptible to oxidative damage (Qin & Rodrigues, 2010b). The specific genetic and biochemical mechanisms responsible for RPE degeneration in AMD

inflammatory responses to necrotic cells (Camara et al., 2011).

**1. Introduction** 


### **Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration**

#### Suofu Qin

*Retinal Disease Research, Department of Biological Sciences, Allergan, Inc., Irvine, CA USA* 

#### **1. Introduction**

24 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

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Inflammation is vital for host defense against invasive pathogens via the recruitment of innate inflammatory cells, which in turn phagocytose infectious agents and produce additional cytokines that then activate adaptive immune responses. Inflammation is also crucial to protect cells and facilitate wound healing as a result of mechanic or chemical injury. Because of the absence of infection, the inflammation induced by metabolic or chemical injury has been termed as 'sterile inflammation' to distinguish from that induced by pathogens. Similar to microbially induced inflammation, the immune system has evolved mechanisms to sense necrotic cell death by responding with innate and adaptive immune response, which is marked by the recruitment of macrophages and the production of inflammatory cytokines. However, unresolved and persistent inflammation due to un-removal or un-containment of the offending agents would turn it into a destructive process that is detrimental to the host. The production of reactive oxygen species (ROS), proteases and inflammatory cytokines causes tissue destruction and fibrosis. Thus, sterile inflammation has been demonstrated to be associated with human diseases such as cardiac ischaemia–reperfusion injury, the restoration of blood flow causes tissue destruction as a result of enhanced production of ROS and inflammatory responses to necrotic cells (Camara et al., 2011).

Sterile inflammation in the etiology of age-related macular degeneration (AMD) has been highlighted by the observations that individuals with genetic mutation in complement factor H confer a significantly higher risky for AMD (Montezuma et al., 2007), an idiopathic retinal degenerative disease that leads to irreversible, profound vision loss in people over 60 year old in developed countries (Evans & Wormald, 1996). AMD occurs in two major forms: atrophic (dry) AMD and exudative (wet) AMD. The atrophic AMD is characterized by RPE atrophy and subjacent photoreceptor degeneration and accounts for approximately 25% of cases with severe central vision loss (Klein et al., 1997). Exudative AMD, which accounts for approximately 75% of cases with severe central vision loss (Klein et al., 1997), is characterized by choroidal neovascularization (CNV) and retinal hemorrhage. These two forms of AMD are both part of the same disease process and share similar risk factors for their development. Although the vision loss results from photoreceptor damage in the central retina, the initial pathogenesis of AMD has been proposed to involve the degeneration of retinal pigment epithelial (RPE) cells (Hageman et al., 2001). The RPE cells *in vivo* has limited regenerating capability upon damage as they are in general post-mitotic and their mitochondria are very susceptible to oxidative damage (Qin & Rodrigues, 2010b). The specific genetic and biochemical mechanisms responsible for RPE degeneration in AMD

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 27

Fig. 1. Cell-surface DAMP receptors that detect a variety of DAMP molecules.

**2.1.1 Nucleic acids** 

Necrotic cells due to environmental and metabolic stress release intracellular damageassociated molecular patterns (DAMPs) or hydrolytic enzymes that degrade extracellular components to generate extracellular DAMPs. Necrotic cells also activate the complement system to generate C3a and C5a. Additionally, oxidized products or oxidized adducts that are strong inflammatory stimuli are also appreciated as DAMP molecules. These DAMPs are sensed by DAMP receptors on host cells, thereby triggering host defense via sterile inflammation. AGEs, advanced glycation-end products; CLRs, c-type lectin receptors; HMGB1, high mobility group box 1; HSPs, heat-shock proteins; oxLDL, oxidized low density proteins; P2X7R, purinergic receptor P2X, ligand-gated ion channel, 7; RAGE, receptor for AGEs; SAP130, spliceosome-associated protein 130; TLRs, toll-like receptors.

Upon releasing from dying or necrotic cells, nucleic acids such as RNA and fragments of genomic DNA can activate innate and adaptive immune systems. Necrotic synovial fluid cells from the patients with rheumatoid arthritis activated fibroblast cells with production of inflammatory cytokines in a toll-like receptor-3 (TLR-3)-dependent manner (Brentano et al., 2005), indicating that the RNA released from necrotic cells is involved in fibroblast cell activation. Heterologous RNA from necrotic cells or *in vitro* transcribed mRNA can activate dendritic cells, which is abolished by RNase pretreatment (Kariko et al., 2004). Moreover, genomic DNA released into cytosol upon cell injury has been found to activate thyroid cells in concert with histone H2B with cytokine production (Kawashima et al., 2011). Mitochondrial DNA released from injured mitochondria can activate neutrophils *in vitro*

have not been determined. However, cumulative oxidative stress and chronic inflammation have been recently appreciated to play important roles in the biogenesis of drusen, the extracellular lipid-containing deposits that are the hallmark of early AMD and may therefore be central to the etiology of this disease (Hageman et al., 2001; Rodrigues, 2007). The eye with its intense exposure to light, robust metabolic activity and high oxygen tension in the macular region, is particularly susceptible to oxidative damage. Thus, there is considerable interest in elucidating the mechanisms responsible for oxidative stress- and sterile inflammation-associated RPE injury, which would provide the basis for designing new strategies to treat or prevent AMD.

Chronic sterile inflammation in the retina might cause RPE cell dysfunction and death that subsequently contribute to retinal degeneration, however, the underlining molecular mechanisms remain elusive. Recently, the damage-associated molecular pattern (DAMP) molecules, a structurally-diverse family of endogenous molecules either released from necrotic cells or breakdown products of the extracellular matrix during cellular injury, are demonstrated to alert host cells for the coming danger by inciting inflammatory responses. Persistent stimulation by the DAMP molecules leads to cell dysfunction and eventually cell death. Some of the DAMP molecules are recognized by pattern recognition receptors, which normally sense pathogen-associated molecular patterns. The role of oxidative stress in the etiology of AMD has been reviewed elsewhere (Qin & Rodrigues, 2010b). In this review, discussed are the nature of the DAMPs, DAMP-initiated inflammatory signaling and the therapeutic potentials of anti-DAMP therapy for AMD intervention.

#### **2. Damage-associated molecular patterns**

The danger hypothesis was first proposed by Matzinger in 1994 to explain how both infectious and non-infectious agents can stimulate adaptive immune responses (Matzinger, 1994). It is postulated that the adaptive immune system has evolved to respond not only to infection but also to non-physiological cell death due to damage or environmental stress. Necrotic cell death is considered as a sign of danger to the organism. According to this danger model, dying cells will release endogenous DAMPs, using similar nomenclature to pathogen-associated molecular patterns (PAMPs). A candidate molecule as a *bona fide* DAMP should meet at least the following three criteria as proposed by Kono and Rock (Kono & Rock, 2008). First, a DAMP should be active as a highly purified molecule rather than owing to endotoxin contamination. Second, the DAMPs should be active at concentrations that are actually present in pathophysiological conditions. Finally, selectively eliminating or inactivating DAMPs will ideally block the biological activity of necrotic cells in *in vitro* and *in vivo* assays (Kono & Rock, 2008). DAMPs are normally sequestered intracellularly and are hidden from recognition by the innate immune system by the plasma membrane under physiological conditions. However, these molecules, when cells undergo injury or necrosis, are released into the extracellular milieu and then trigger inflammation under sterile conditions. Based on their origin, DAMPs are classified into two categories: intracellular and extracellular DAMPs.

#### **2.1 Intracellular DAMPs**

Intracellular DAMPs are bioactive mediators of intracellular origin that directly stimulate cells of the innate system. They are pre-existed within the cells and released into extracellular environment after cell injury or death. The DAMPs associated with retinal pathogenesis are summarized in Figure 1.

Fig. 1. Cell-surface DAMP receptors that detect a variety of DAMP molecules.

Necrotic cells due to environmental and metabolic stress release intracellular damageassociated molecular patterns (DAMPs) or hydrolytic enzymes that degrade extracellular components to generate extracellular DAMPs. Necrotic cells also activate the complement system to generate C3a and C5a. Additionally, oxidized products or oxidized adducts that are strong inflammatory stimuli are also appreciated as DAMP molecules. These DAMPs are sensed by DAMP receptors on host cells, thereby triggering host defense via sterile inflammation. AGEs, advanced glycation-end products; CLRs, c-type lectin receptors; HMGB1, high mobility group box 1; HSPs, heat-shock proteins; oxLDL, oxidized low density proteins; P2X7R, purinergic receptor P2X, ligand-gated ion channel, 7; RAGE, receptor for AGEs; SAP130, spliceosome-associated protein 130; TLRs, toll-like receptors.

#### **2.1.1 Nucleic acids**

26 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

have not been determined. However, cumulative oxidative stress and chronic inflammation have been recently appreciated to play important roles in the biogenesis of drusen, the extracellular lipid-containing deposits that are the hallmark of early AMD and may therefore be central to the etiology of this disease (Hageman et al., 2001; Rodrigues, 2007). The eye with its intense exposure to light, robust metabolic activity and high oxygen tension in the macular region, is particularly susceptible to oxidative damage. Thus, there is considerable interest in elucidating the mechanisms responsible for oxidative stress- and sterile inflammation-associated RPE injury, which would provide the basis for designing

Chronic sterile inflammation in the retina might cause RPE cell dysfunction and death that subsequently contribute to retinal degeneration, however, the underlining molecular mechanisms remain elusive. Recently, the damage-associated molecular pattern (DAMP) molecules, a structurally-diverse family of endogenous molecules either released from necrotic cells or breakdown products of the extracellular matrix during cellular injury, are demonstrated to alert host cells for the coming danger by inciting inflammatory responses. Persistent stimulation by the DAMP molecules leads to cell dysfunction and eventually cell death. Some of the DAMP molecules are recognized by pattern recognition receptors, which normally sense pathogen-associated molecular patterns. The role of oxidative stress in the etiology of AMD has been reviewed elsewhere (Qin & Rodrigues, 2010b). In this review, discussed are the nature of the DAMPs, DAMP-initiated inflammatory signaling and the

The danger hypothesis was first proposed by Matzinger in 1994 to explain how both infectious and non-infectious agents can stimulate adaptive immune responses (Matzinger, 1994). It is postulated that the adaptive immune system has evolved to respond not only to infection but also to non-physiological cell death due to damage or environmental stress. Necrotic cell death is considered as a sign of danger to the organism. According to this danger model, dying cells will release endogenous DAMPs, using similar nomenclature to pathogen-associated molecular patterns (PAMPs). A candidate molecule as a *bona fide* DAMP should meet at least the following three criteria as proposed by Kono and Rock (Kono & Rock, 2008). First, a DAMP should be active as a highly purified molecule rather than owing to endotoxin contamination. Second, the DAMPs should be active at concentrations that are actually present in pathophysiological conditions. Finally, selectively eliminating or inactivating DAMPs will ideally block the biological activity of necrotic cells in *in vitro* and *in vivo* assays (Kono & Rock, 2008). DAMPs are normally sequestered intracellularly and are hidden from recognition by the innate immune system by the plasma membrane under physiological conditions. However, these molecules, when cells undergo injury or necrosis, are released into the extracellular milieu and then trigger inflammation under sterile conditions. Based on their origin, DAMPs are classified into two categories:

Intracellular DAMPs are bioactive mediators of intracellular origin that directly stimulate cells of the innate system. They are pre-existed within the cells and released into extracellular environment after cell injury or death. The DAMPs associated with retinal

therapeutic potentials of anti-DAMP therapy for AMD intervention.

**2. Damage-associated molecular patterns** 

intracellular and extracellular DAMPs.

pathogenesis are summarized in Figure 1.

**2.1 Intracellular DAMPs** 

new strategies to treat or prevent AMD.

Upon releasing from dying or necrotic cells, nucleic acids such as RNA and fragments of genomic DNA can activate innate and adaptive immune systems. Necrotic synovial fluid cells from the patients with rheumatoid arthritis activated fibroblast cells with production of inflammatory cytokines in a toll-like receptor-3 (TLR-3)-dependent manner (Brentano et al., 2005), indicating that the RNA released from necrotic cells is involved in fibroblast cell activation. Heterologous RNA from necrotic cells or *in vitro* transcribed mRNA can activate dendritic cells, which is abolished by RNase pretreatment (Kariko et al., 2004). Moreover, genomic DNA released into cytosol upon cell injury has been found to activate thyroid cells in concert with histone H2B with cytokine production (Kawashima et al., 2011). Mitochondrial DNA released from injured mitochondria can activate neutrophils *in vitro*

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 29

(Scaffidi et al., 2002). HMGB1 release has been detected from retinal cell death by oxidative stress *in vitro* and retinal detachment *in vivo* (Arimura et al., 2009). The increase in the vitreous HMGB1 level is correlated with that of monocyte chemotactic protein-1 (MCP-1) in

HSPs are a highly conserved group of intracellular proteins classified into HSP110, HSP90, HSP70, HSP60, and small molecular HSPs based on their molecular weights, and function as molecular chaperones to promote the refolding of damaged proteins and inhibit protein aggregation under stress conditions (Georgopoulos & Welch, 1993). Purified HSP70 stimulates activation of NF-*k*B in monocytes with production of inflammatory cytokines (Asea et al., 2000) and transgenic expression of HSP70 enhances the extent of *in vivo* sterile inflammation upon β-cell damage (Alam et al., 2009). HSPs also can function as a chaperone to target the antigenic peptides to antigen-presenting cells, thereby initiating immune responses (Binder et al., 2007). Dying cells express higher levels of HSPs (Decanini et al., 2007), thereby providing danger signals to alert the neighboring cells for upcoming danger. However, caution should be

exercised as it is still controversial whether extracellular HSPs function as cytokines.

Whether S100 proteins contribute to disease pathogenesis remains to be confirmed.

The S100 proteins are a family of about 20 related small, acidic calcium-binding proteins that modulate an array of intracellular functions, like calcium homeostasis, cell cycle and cytoskeletal organization (Heizmann et al., 2002). S100 proteins are higher in extracellular milieu at inflammation site. S100A8 and/or S100A9 stimulate migration of neutrophils and monocytes in gouty arthritis, which is inhibited by anti-S100 antibodies (Ryckman et al., 2003). Additionally, S100B induces cell death in cultured RPE cells (Howes et al., 2004).

Extracellular DAMPs, such as hyaluronan, heparan sulphate and biglycan, are generated as a result of proteolysis by enzymes released from dying cells or by proteases activated to promote tissue repair and remodeling (Babelova et al., 2009). Extracellular DAMPs can also be generated from activation of complement system by the degraded or released molecules

Extracellular matrix components, which are thought to function as structural elements, are now gaining recognition as signaling molecules triggering or enhancing sterile inflammation once cleaved by released proteolytic enzymes during tissue injury. Hyaluronan fragments generated upon cell injury activate endothelial cells *in vitro* and *in vivo* with significant production of chemokine interleukin-8 (IL-8) (Taylor et al., 2004) and induce maturation of dendritic cells (Termeer et al., 2002). In addition, biglycan has been shown to activate macrophages accompanied with NF-κB-dependent cytokine production (Schaefer et al., 2005). Activated macrophages also release biglycan, further amplifying inflammatory responses (Schaefer et al., 2005). Biglycan can stimulate synthesis of immature

human eyes with retinal degeneration.

**2.1.5 Heat-shock proteins (HSPs)** 

**2.1.6 S100 proteins** 

**2.2 Extracellular DAMPs** 

from necrotic cells (Garg et al., 2010).

**2.2.1 Breakdown products of extracellular matrix** 

and *in vivo*, eliciting neutrophil-mediated tissue injury (Zhang et al., 2010). Intriguingly, *Alu* RNA, a double-stranded RNA (dsRNA) isolated from drusen of the patients with geographic atrophy can cause RPE cell death *in vitro* and RPE layer degeneration *in vivo* (Kaneko et al., 2011). Knockout of TLR3, the receptor for RNA, protects necrosis-induced retinal degeneration in mouse (Shiose et al., 2011). These results implicate that released nucleic acids from necrotic cells might have a role in etiology of AMD.

#### **2.1.2 Interlukin-1**α **(IL-1**α**)**

IL-1α is synthesized as a biologically active cytokine but is retained in cytosol and nucleus under physiological conditions (Cohen et al., 2010). However, IL-1α is released with its cellular contents when cells undergo necrosis and released IL-1α activates its cognate receptor, leading to rapid recruitment of inflammatory cells into the surrounding injured tissue (Cohen et al., 2010). IL-1α in dying cells and functional IL-1R are required for neutrophilic response to dead cells and tissue injury *in vivo* while this pathway is not essential for the neutrophil response to a microbial stimulus (Chen et al., 2007). Role of IL-1α in sterile inflammation appears to be dependent on its sources. IL-1α released from necrotic cells primarily triggers initial neutrohphil response and primes resident macrophage that produces IL-1α, required for necrosis-induced sterile inflammation (Kono et al., 2010b). Interestingly, IL-1α from necrotic dendritic cells primes mesothelial cells that generate chemokine (C-X-C motif) ligand 1, then recruiting neutrophils into sterile inflammation sites (Eigenbrod et al., 2008).

#### **2.1.3 ATP and uric acid**

The cytoplasm of each cell contains high concentrations of ATP, however, extracellular levels are quite low as ATP is quickly degraded by ecto-ATPases in normal tissues (Di Virgilio, 2007). Upon cell damage due to chemical or mechanical injury, ATP levels in extracellular environment is increased rapidly. High levels of ATP in extracellular space have been observed during airway inflammation *in vivo* (Idzko et al., 2007). The increase in extracellular ATP concentrations subsequently triggers inflammatory responses since lowering ATP levels by apyrase abolishes cardinal features of asthma such as cytokine production (Idzko et al., 2007). Addition of ATP to cell culture results in significant release/production of inflammatory mediators and causes cell death if under persistent stimulation (Surprenant et al., 1996). Stimulation of RPE cells with ATP enhances cytokine production (Relvas et al., 2009) and then RPE cell death (Yang et al., 2010). Uric acid, a ubiquitous metabolite of purine-degradation pathway, can be produced in high quantities upon cellular injury (Kono et al., 2010a). Uric acid, presented as monosodium urate (MSU) crystals in salt-rich fluids, promotes acute inflammatory responses *in vivo* which is substantially inhibited by uric acid depletion (Kono et al., 2010a).

#### **2.1.4 High-mobility group box 1 protein (HMGB1)**

HMGB1 is a chromatin-binding protein with key role in nuclear homeostasis. HMGB1 in extracellular mellitus behaves as a cytokine, promoting inflammation and disease pathogenesis. HMGB1 was first identified to mediate endotoxin-induced lethality in mouse (Wang et al., 1999). Addition of purified recombinant HMGB1 stimulates production of inflammatory cytokines in human monocytes (Andersson et al., 2000) and knockout of HMGB1 significantly inhibits the capability of necrotic cells to promote inflammation (Scaffidi et al., 2002). HMGB1 release has been detected from retinal cell death by oxidative stress *in vitro* and retinal detachment *in vivo* (Arimura et al., 2009). The increase in the vitreous HMGB1 level is correlated with that of monocyte chemotactic protein-1 (MCP-1) in human eyes with retinal degeneration.

#### **2.1.5 Heat-shock proteins (HSPs)**

28 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

and *in vivo*, eliciting neutrophil-mediated tissue injury (Zhang et al., 2010). Intriguingly, *Alu* RNA, a double-stranded RNA (dsRNA) isolated from drusen of the patients with geographic atrophy can cause RPE cell death *in vitro* and RPE layer degeneration *in vivo* (Kaneko et al., 2011). Knockout of TLR3, the receptor for RNA, protects necrosis-induced retinal degeneration in mouse (Shiose et al., 2011). These results implicate that released

IL-1α is synthesized as a biologically active cytokine but is retained in cytosol and nucleus under physiological conditions (Cohen et al., 2010). However, IL-1α is released with its cellular contents when cells undergo necrosis and released IL-1α activates its cognate receptor, leading to rapid recruitment of inflammatory cells into the surrounding injured tissue (Cohen et al., 2010). IL-1α in dying cells and functional IL-1R are required for neutrophilic response to dead cells and tissue injury *in vivo* while this pathway is not essential for the neutrophil response to a microbial stimulus (Chen et al., 2007). Role of IL-1α in sterile inflammation appears to be dependent on its sources. IL-1α released from necrotic cells primarily triggers initial neutrohphil response and primes resident macrophage that produces IL-1α, required for necrosis-induced sterile inflammation (Kono et al., 2010b). Interestingly, IL-1α from necrotic dendritic cells primes mesothelial cells that generate chemokine (C-X-C motif) ligand 1, then recruiting neutrophils into sterile inflammation sites

The cytoplasm of each cell contains high concentrations of ATP, however, extracellular levels are quite low as ATP is quickly degraded by ecto-ATPases in normal tissues (Di Virgilio, 2007). Upon cell damage due to chemical or mechanical injury, ATP levels in extracellular environment is increased rapidly. High levels of ATP in extracellular space have been observed during airway inflammation *in vivo* (Idzko et al., 2007). The increase in extracellular ATP concentrations subsequently triggers inflammatory responses since lowering ATP levels by apyrase abolishes cardinal features of asthma such as cytokine production (Idzko et al., 2007). Addition of ATP to cell culture results in significant release/production of inflammatory mediators and causes cell death if under persistent stimulation (Surprenant et al., 1996). Stimulation of RPE cells with ATP enhances cytokine production (Relvas et al., 2009) and then RPE cell death (Yang et al., 2010). Uric acid, a ubiquitous metabolite of purine-degradation pathway, can be produced in high quantities upon cellular injury (Kono et al., 2010a). Uric acid, presented as monosodium urate (MSU) crystals in salt-rich fluids, promotes acute inflammatory responses *in vivo* which is

HMGB1 is a chromatin-binding protein with key role in nuclear homeostasis. HMGB1 in extracellular mellitus behaves as a cytokine, promoting inflammation and disease pathogenesis. HMGB1 was first identified to mediate endotoxin-induced lethality in mouse (Wang et al., 1999). Addition of purified recombinant HMGB1 stimulates production of inflammatory cytokines in human monocytes (Andersson et al., 2000) and knockout of HMGB1 significantly inhibits the capability of necrotic cells to promote inflammation

nucleic acids from necrotic cells might have a role in etiology of AMD.

substantially inhibited by uric acid depletion (Kono et al., 2010a).

**2.1.4 High-mobility group box 1 protein (HMGB1)** 

**2.1.2 Interlukin-1**α **(IL-1**α**)** 

(Eigenbrod et al., 2008).

**2.1.3 ATP and uric acid** 

HSPs are a highly conserved group of intracellular proteins classified into HSP110, HSP90, HSP70, HSP60, and small molecular HSPs based on their molecular weights, and function as molecular chaperones to promote the refolding of damaged proteins and inhibit protein aggregation under stress conditions (Georgopoulos & Welch, 1993). Purified HSP70 stimulates activation of NF-*k*B in monocytes with production of inflammatory cytokines (Asea et al., 2000) and transgenic expression of HSP70 enhances the extent of *in vivo* sterile inflammation upon β-cell damage (Alam et al., 2009). HSPs also can function as a chaperone to target the antigenic peptides to antigen-presenting cells, thereby initiating immune responses (Binder et al., 2007). Dying cells express higher levels of HSPs (Decanini et al., 2007), thereby providing danger signals to alert the neighboring cells for upcoming danger. However, caution should be exercised as it is still controversial whether extracellular HSPs function as cytokines.

#### **2.1.6 S100 proteins**

The S100 proteins are a family of about 20 related small, acidic calcium-binding proteins that modulate an array of intracellular functions, like calcium homeostasis, cell cycle and cytoskeletal organization (Heizmann et al., 2002). S100 proteins are higher in extracellular milieu at inflammation site. S100A8 and/or S100A9 stimulate migration of neutrophils and monocytes in gouty arthritis, which is inhibited by anti-S100 antibodies (Ryckman et al., 2003). Additionally, S100B induces cell death in cultured RPE cells (Howes et al., 2004). Whether S100 proteins contribute to disease pathogenesis remains to be confirmed.

#### **2.2 Extracellular DAMPs**

Extracellular DAMPs, such as hyaluronan, heparan sulphate and biglycan, are generated as a result of proteolysis by enzymes released from dying cells or by proteases activated to promote tissue repair and remodeling (Babelova et al., 2009). Extracellular DAMPs can also be generated from activation of complement system by the degraded or released molecules from necrotic cells (Garg et al., 2010).

#### **2.2.1 Breakdown products of extracellular matrix**

Extracellular matrix components, which are thought to function as structural elements, are now gaining recognition as signaling molecules triggering or enhancing sterile inflammation once cleaved by released proteolytic enzymes during tissue injury. Hyaluronan fragments generated upon cell injury activate endothelial cells *in vitro* and *in vivo* with significant production of chemokine interleukin-8 (IL-8) (Taylor et al., 2004) and induce maturation of dendritic cells (Termeer et al., 2002). In addition, biglycan has been shown to activate macrophages accompanied with NF-κB-dependent cytokine production (Schaefer et al., 2005). Activated macrophages also release biglycan, further amplifying inflammatory responses (Schaefer et al., 2005). Biglycan can stimulate synthesis of immature

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 31

2003). With the accumulation of AGEs, receptor for AGEs (RAGE) is simultaneously induced (Yamada et al., 2006), further amplifying cellular activation. Induction of AGE formation *in vivo* leads to the increased transcription of inflammatory genes, resulting in ageing of the RPE/choroid (Tian et al., 2005). In cultured human RPE cells, activation of AGE-RAGE pathway stimulates expression of VEGF (Ma et al., 2007), and production of IL-8 and MCP-1 (Bian et al., 2001). Moreover, activation of RAGE can trigger RPE cell death *in vitro* (Howes et

Lipid peroxidation, triggered by direct photobleaching of PUFAs or indirect excitation of photosensitizers contained in RPE lipofuscin, generates a number of reactive dicarbonyl compounds (aldehydes) such as acrolein, 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA) and CEP (Glenn & Stitt, 2009). These aldehydes can deplete cellular thiols, resulting in cell death. Direct exposure of RPE cells to 4-HNE causes dysregulation of chemokine production, increase in cell permeability, and finally cell death (Qin & Rodrigues, 2010a). Moreover, these aldehydes can alter cell functions via formation of lipid adducts on free amino groups of proteins. Among them, CEP adducts, uniquely generated by the oxidation of the most oxidizable fatty acid docosahexaenoate in human retina (Gu et al., 2003), are the most abundant class of oxidized proteins found in drusen. CEP adducts are higher in photoreceptors from AMD patients than healthy retinas (Gu et al., 2003). Mice immunized with mouse serum albumin (MSA) adducted with CEP (CEP-MSA) develop an atrophic AMD-like phenotype including RPE loss and drusen formation with accumulation of macrophages in the interphotoreceptor matrix and C3 fragments in Bruch's membrane (Hollyfield et al., 2008). CEP-MSA also stimulates angiogenesis in ex-vivo models and subretinal injection of CEP-MSA exacerbates laser-induced CNV in mice (Ebrahem et al., 2006). These observations definitely implicate oxidized lipid adducts in the perturbation of

Low-density lipoproteins (LDL) are complex particles containing cholesterol, phosphlipids, and triglycerides. The PUFAs in those molecules are susceptible to free radical-initiated oxidation, generating chemically-reactive such as MDA and 4-HNE and bioactive molecules. MDA and 4-HNE oxidize proteins, forming MDA lysine or 4-HNE cysteine protein adducts that are the major modifications observed in RPE lipofuscin (Schutt et al., 2003). OxLDL is found to be accumulated in AMD lesions (Kamei et al., 2007) and higher in patients' blood with AMD (Javadzadeh et al., 2010). Oxidation of the cholesterol within the LDL particle generates a series of cholesterol oxides, of which 7-ketocholesterol is very toxic to RPE cells (Moreira et al., 2009). Exposure of RPE cells to oxLDL inhibits POS phagocytosis, an important RPE cell function essential for outer segment renewal and survival of photoreceptors, by altering phagosome maturation (Hoppe et al., 2004b) and mis-sorting the principal lysosomal protease cathepsin D (Hoppe et al., 2004a). Moreover, oxLDL induces transcriptional alterations in genes related to lipid metabolism, oxidative stress, inflammation and apoptosis in RPE cells (Yamada et al., 2008; Yu et al., 2009). OxLDL causes RPE cell death, at least in part, through formation of 7-ketocholesterol (Rodriguez et al., 2004). Additionally, oxLDL is ligand for scavenger receptors expressed on macrophages that are recruited to the subretinal sites where oxLDL accumulates, further stressing RPE cells via amplifying inflammatory responses

al., 2004), demonstrating that AGEs are toxic to retinal cells.

RPE cell function, leading RPE cell death both *in vitro* and *in vivo*.

**2.3.3 Oxidized low-density lipoproteins (oxLDL)** 

**2.3.2 Carboxyethyl pyrole (CEP) adducts** 

IL-1β via toll-like receptor signaling and in the mean time promote the processing of immature IL-1β to its mature form through P2X receptor signaling (Babelova et al., 2009). Importantly, elevation of hyaluronan contributes to the development of laser-induced choroidal neovascularization with recruitment of macrophages to the lesion sites (Mochimaru et al., 2009), shedding light on understanding the roles of extracellular components in etiology of AMD.

#### **2.2.2 C3a and C5a**

In the retina, photooxidation causes oxidative stress and complement activation, leading to cell death *in vitro* and *in vivo* (Radu et al., 2011; Zhou et al., 2006). Thus, the chance of RPE/photoreceptor cells being attacked by activated complement systems is increased. During the process of complement cascade activation, the cleaved complement components C3a, C4a and C5a, known as anaphylotoxins, stimulate inflammation. In cultured RPE cells, treatment with C5a stimulates production of IL-8 (Fukuoka et al., 2003) and MCP-1 (Ambati et al., 2003). Furthermore, C3a and C5a have been shown to be present in drusen (Ambati et al., 2003) and are generated early in the course of laser-induced CNV where activation of C3aR or C5aR is required for CNV formation (Nozaki et al., 2006), supporting the idea that RPE cells are constantly stimulated by C3a and C5a and complement–driven sterile inflammation is involved in the etiology and progression of AMD.

#### **2.3 Oxidized adducts**

With its unusually abundance in poly-unsaturated fatty acids (PUFAs), glucose-enriched and oxidative environment, the retina is an ideal place to form free radicals and bioactive small molecules, then oxidizing proteins, lipids and DNA. Many oxidized adducts of proteins with lipid or glucose accumulate within and around RPE/photoreceptor cells as a function of ageing. Accumulation of these oxidized adducts triggers transcriptional alterations in genes related to cell death and inflammatory response, perturbs the lysosomal function of the RPE via delayed processing of photoreceptor outer segments (POS), thereby resulting in the disease pathogenesis. Although they are not necessarily associated with necrosis, oxidized adducts also generate pattern recognition sites such as oxidized phospholipids, oxidized lipoproteins and long-chain fatty acids that are strong sterile stimuli. These oxidized adducts play important roles in sterile inflammation and potentially in the etiology of human diseases so that they should be recognized as DAMP molecules. Discussed here are four examples of oxidized adducts, advanced glycation endproducts (AGEs), carboxyethyl pyrole (CEP)-protein adducts, oxidized low-density lipoproteins (oxLDL) and oxidized bis-retinoid pyridinium (A2E) with relevance to AMD etiology.

#### **2.3.1 Advanced glycation end-products (AGEs)**

AGEs are heterogeneous non-enzymatic glycation products of proteins, lipids and DNA on free amino groups by aldehyde groups on sugars. AGEs accumulate during normal ageing with their formation being accelerated in a setting of oxidative stress and inflammation (Schleicher et al., 1997). There is little or no AGE products detected in normal retina, but expression of AGE products increases concomitantly with drusen formation and development of early AMD (Howes et al., 2004). AGE products are also present in RPE lipofuscin, an enzymatically undegradable heterogeneous mixture of numerous biomolecules (Schutt et al., 2003). With the accumulation of AGEs, receptor for AGEs (RAGE) is simultaneously induced (Yamada et al., 2006), further amplifying cellular activation. Induction of AGE formation *in vivo* leads to the increased transcription of inflammatory genes, resulting in ageing of the RPE/choroid (Tian et al., 2005). In cultured human RPE cells, activation of AGE-RAGE pathway stimulates expression of VEGF (Ma et al., 2007), and production of IL-8 and MCP-1 (Bian et al., 2001). Moreover, activation of RAGE can trigger RPE cell death *in vitro* (Howes et al., 2004), demonstrating that AGEs are toxic to retinal cells.

#### **2.3.2 Carboxyethyl pyrole (CEP) adducts**

30 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

IL-1β via toll-like receptor signaling and in the mean time promote the processing of immature IL-1β to its mature form through P2X receptor signaling (Babelova et al., 2009). Importantly, elevation of hyaluronan contributes to the development of laser-induced choroidal neovascularization with recruitment of macrophages to the lesion sites (Mochimaru et al., 2009), shedding light on understanding the roles of extracellular

In the retina, photooxidation causes oxidative stress and complement activation, leading to cell death *in vitro* and *in vivo* (Radu et al., 2011; Zhou et al., 2006). Thus, the chance of RPE/photoreceptor cells being attacked by activated complement systems is increased. During the process of complement cascade activation, the cleaved complement components C3a, C4a and C5a, known as anaphylotoxins, stimulate inflammation. In cultured RPE cells, treatment with C5a stimulates production of IL-8 (Fukuoka et al., 2003) and MCP-1 (Ambati et al., 2003). Furthermore, C3a and C5a have been shown to be present in drusen (Ambati et al., 2003) and are generated early in the course of laser-induced CNV where activation of C3aR or C5aR is required for CNV formation (Nozaki et al., 2006), supporting the idea that RPE cells are constantly stimulated by C3a and C5a and complement–driven sterile

With its unusually abundance in poly-unsaturated fatty acids (PUFAs), glucose-enriched and oxidative environment, the retina is an ideal place to form free radicals and bioactive small molecules, then oxidizing proteins, lipids and DNA. Many oxidized adducts of proteins with lipid or glucose accumulate within and around RPE/photoreceptor cells as a function of ageing. Accumulation of these oxidized adducts triggers transcriptional alterations in genes related to cell death and inflammatory response, perturbs the lysosomal function of the RPE via delayed processing of photoreceptor outer segments (POS), thereby resulting in the disease pathogenesis. Although they are not necessarily associated with necrosis, oxidized adducts also generate pattern recognition sites such as oxidized phospholipids, oxidized lipoproteins and long-chain fatty acids that are strong sterile stimuli. These oxidized adducts play important roles in sterile inflammation and potentially in the etiology of human diseases so that they should be recognized as DAMP molecules. Discussed here are four examples of oxidized adducts, advanced glycation endproducts (AGEs), carboxyethyl pyrole (CEP)-protein adducts, oxidized low-density lipoproteins (oxLDL) and oxidized bis-retinoid pyridinium (A2E) with relevance to AMD etiology.

AGEs are heterogeneous non-enzymatic glycation products of proteins, lipids and DNA on free amino groups by aldehyde groups on sugars. AGEs accumulate during normal ageing with their formation being accelerated in a setting of oxidative stress and inflammation (Schleicher et al., 1997). There is little or no AGE products detected in normal retina, but expression of AGE products increases concomitantly with drusen formation and development of early AMD (Howes et al., 2004). AGE products are also present in RPE lipofuscin, an enzymatically undegradable heterogeneous mixture of numerous biomolecules (Schutt et al.,

inflammation is involved in the etiology and progression of AMD.

**2.3.1 Advanced glycation end-products (AGEs)** 

components in etiology of AMD.

**2.2.2 C3a and C5a** 

**2.3 Oxidized adducts** 

Lipid peroxidation, triggered by direct photobleaching of PUFAs or indirect excitation of photosensitizers contained in RPE lipofuscin, generates a number of reactive dicarbonyl compounds (aldehydes) such as acrolein, 4-hydroxy-2-nonenal (4-HNE), malondialdehyde (MDA) and CEP (Glenn & Stitt, 2009). These aldehydes can deplete cellular thiols, resulting in cell death. Direct exposure of RPE cells to 4-HNE causes dysregulation of chemokine production, increase in cell permeability, and finally cell death (Qin & Rodrigues, 2010a). Moreover, these aldehydes can alter cell functions via formation of lipid adducts on free amino groups of proteins. Among them, CEP adducts, uniquely generated by the oxidation of the most oxidizable fatty acid docosahexaenoate in human retina (Gu et al., 2003), are the most abundant class of oxidized proteins found in drusen. CEP adducts are higher in photoreceptors from AMD patients than healthy retinas (Gu et al., 2003). Mice immunized with mouse serum albumin (MSA) adducted with CEP (CEP-MSA) develop an atrophic AMD-like phenotype including RPE loss and drusen formation with accumulation of macrophages in the interphotoreceptor matrix and C3 fragments in Bruch's membrane (Hollyfield et al., 2008). CEP-MSA also stimulates angiogenesis in ex-vivo models and subretinal injection of CEP-MSA exacerbates laser-induced CNV in mice (Ebrahem et al., 2006). These observations definitely implicate oxidized lipid adducts in the perturbation of RPE cell function, leading RPE cell death both *in vitro* and *in vivo*.

#### **2.3.3 Oxidized low-density lipoproteins (oxLDL)**

Low-density lipoproteins (LDL) are complex particles containing cholesterol, phosphlipids, and triglycerides. The PUFAs in those molecules are susceptible to free radical-initiated oxidation, generating chemically-reactive such as MDA and 4-HNE and bioactive molecules. MDA and 4-HNE oxidize proteins, forming MDA lysine or 4-HNE cysteine protein adducts that are the major modifications observed in RPE lipofuscin (Schutt et al., 2003). OxLDL is found to be accumulated in AMD lesions (Kamei et al., 2007) and higher in patients' blood with AMD (Javadzadeh et al., 2010). Oxidation of the cholesterol within the LDL particle generates a series of cholesterol oxides, of which 7-ketocholesterol is very toxic to RPE cells (Moreira et al., 2009). Exposure of RPE cells to oxLDL inhibits POS phagocytosis, an important RPE cell function essential for outer segment renewal and survival of photoreceptors, by altering phagosome maturation (Hoppe et al., 2004b) and mis-sorting the principal lysosomal protease cathepsin D (Hoppe et al., 2004a). Moreover, oxLDL induces transcriptional alterations in genes related to lipid metabolism, oxidative stress, inflammation and apoptosis in RPE cells (Yamada et al., 2008; Yu et al., 2009). OxLDL causes RPE cell death, at least in part, through formation of 7-ketocholesterol (Rodriguez et al., 2004). Additionally, oxLDL is ligand for scavenger receptors expressed on macrophages that are recruited to the subretinal sites where oxLDL accumulates, further stressing RPE cells via amplifying inflammatory responses

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 33

necrotic cells while TLR9 senses endogenous DNA (Zhang et al., 2010), triggering sterile inflammatory response and subsequent toxicity. TLR3 is shown to mediate retinal degeneration caused by impaired clearance of toxic all-trans retinal in mice since TLR3 deficiency confers retina protection (Shiose et al., 2011). Choroidal neovascular membranes from AMD patients expressed higher levels of TLR3 in RPE cells (Maloney et al., 2010) and TLR3 activation by siRNA inhibits CNV as siRNA inhibition is abolished in TLR3-deficient mice (Kleinman et al., 2008). Moreover, dsRNA causes RPE cell death that is mediated by TLR3 and genetic variant in the TLR3 412Phe confers protection against geographic atrophy

CLRs including DEC205, Mincle, CLEC9A and DC-SIGN are a family of surface receptors known to recognize carbohydrate moieties on viruses, bacteria and fungi (Cambi & Figdor, 2009). Stimulation of CLRs leads to activation of signaling pathways that elevate cytokine production. Although their ligands are poorly defined, Mincle (also known as CLEC4E) and CLEC9A can sense necrotic cell death (Cambi & Figdor, 2009). Mincle recognizes SAP130 from necrotic cells and triggers intracellular signaling via the associated FcRγ adaptor, leading to the production of inflammatory cytokines (Yamasaki et al., 2008). CLEC9A, a Sykcoupled CLR, can recognize necrotic cells and present dead cell-associated antigens to CD8+ T cells (Sancho et al., 2009). Similar to Mincle, the capability of CLEC9A to recognize necrotic cells makes it a potential receptor that is important for sterile inflammatory

NLRs, consisting of the three subfamilies with 14 NALPs, 6 NODs and 2 IPAF/NAIP, are cytosolic PRRs that sense microbial invasion, eliciting an inflammatory response to alert the system to the presence of danger, mainly by assembling inflammasomes that activate caspase-1 for processing immature IL-1β to mature IL-1β (Martinon et al., 2009). NLRs contain a central nucleotide-binding oligomerization domain (NACHT), an N-terminal effector domain (pyrin domain, caspase-recruitment domain or BIR domain) and C-terminal leucine-rich repeats (LRRs). Among the NLRs identified so far, the NOD-, LRR-, and pyrin domain-containing 3 (NLRP3, also termed as NALP3) has been shown to be capable of

AIM2 is a cytosolic protein containing a C-terminal HIN200 and an N-terminal PYD domain, which is identified to recognize dsDNA derived from virus and bacterial, triggering anti-virus responses (Burckstummer et al., 2009). AIM2 can also be activated by the transfection of synthetic dsDNA (Fernandes-Alnemri et al., 2009), highlighting that the innate response to DNA is regulated by the localization of DNA in concert with the innate receptors rather than the source of DNA. Under physiological conditions, self-DNA is localized in nuclei and mitochondria. However, injured or dying cells can release mitochondrial and genomic DNA into the cytosol where AIM2 resides. Released genomic and mitochondrial DNA have been demonstrated to cause inflammatory responses (Kawashima et al., 2011; Zhang et al., 2010). Whether there is a pathogenic role for AIM2 in

detecting endogenous danger signals (*See* discussion in NLRP3 inflammasome).

(Yang et al., 2008). These observations reveal a role of TLR3 in AMD development.

**3.1.2 C-type lectin receptors (CLRs)** 

**3.1.3 NOD-like receptors (NLRs)** 

**3.1.4 Absence in melanoma 2 (AIM2)** 

sterile inflammation-related diseases remains unclear.

responses even though its ligands are unidentified.

(Kamei et al., 2007). Collectively, these observations suggest a causal role of oxLDL accumulation in AMD pathogenesis although the precise mechanisms are not well defined

#### **2.3.4 Bis-retinoid pyridinium A2E**

A2E, a byproduct of the visual cycle, is formed through the condensation of two molecules of all-trans-retinal with one molecule of phosphatidylethanolamine in the POS upon photoisomerization of 11-*cis* retinal (Mata et al., 2000). A2E accumulates as lipofuscin in RPE cells with ageing since it is resistant to enzymatic degradation. After light, in particular blue light irradiation, A2E is oxidized, initially by addition of light-excited singlet oxygen, and oxidized A2E can then generate free radicals such as superoxide anion and hydroxyl radical, or discompose to reactive dicarbonyls like methylglyoxal, triggering free radical chain reaction (Wu et al., 2010). Light exposure causes death of A2E-loaded RPE cells in other hand A2E-free RPE cells are not (Schutt et al., 2000). Moreover, photooxidation products of A2E have been shown to activate complement system in RPE cells (Zhou et al., 2006), suggesting that sterile inflammation is involved in A2E cytotoxicity. However, a causal role of A2E accumulation in AMD development remains to be confirmed.

#### **3. DAMP receptors**

How does the innate immune system distinguish between dead and live cells? The crucial event upon necrotic cell death is the release of intracellular DAMPs and generation of extracellular DAMPs which can be sensed by the receptors on innate immune cells. This section discusses the recent progress in the recognition of DAMPs by pattern recognition receptors and DAMP receptors that are not typically associated with microbial recognition (Figure 1).

#### **3.1 Pattern recognition receptors**

Pattern recognition receptors (PRRs) recognize conserved structural moieties called PAMPs found in microorganisms and in turn induce cytokine production, which is important in inflammatory and antimicrobial responses. Up to date, five classes of PRRs have been identified. They are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), the retinoic acidinducible gene (RIG)-I-like receptors (RLRs) and absence in melanoma 2 (AIM2). The membrane-bound TLRs and CLRs surveillance the extracellular milieu, whereas RLRs, NLRs and AIM2-like receptors have emerged as pivotal sensors of infection and stress in intracellular compartments (Meylan et al., 2006). Among them, TLRs, CLRs, NLRs and AIM2 participate in DAMP-dependent inflammatory responses.

#### **3.1.1 Toll-like receptors (TLRs)**

TLRs have been reported to be activated by intracellular DAMPs including HSPs, S100 proteins, uric acid and HMGB1, as well as extracellular DAMPs such as hyaluronan, heparan sulphate and proteoglycans (Kono & Rock, 2008). RPE cells make up the first line of defense against pathogens by expressing almost all TLR iso-forms except TLR-8 (Kumar et al., 2004). Activation of TLR3 and TLR9 leads to the production of inflammatory mediators including cytokines and adhesion molecules in cultured RPE cells (Ebihara et al., 2007). TLR3 detects mRNA (Kariko et al., 2004) and dsRNA (Dogusan et al., 2008) released from necrotic cells while TLR9 senses endogenous DNA (Zhang et al., 2010), triggering sterile inflammatory response and subsequent toxicity. TLR3 is shown to mediate retinal degeneration caused by impaired clearance of toxic all-trans retinal in mice since TLR3 deficiency confers retina protection (Shiose et al., 2011). Choroidal neovascular membranes from AMD patients expressed higher levels of TLR3 in RPE cells (Maloney et al., 2010) and TLR3 activation by siRNA inhibits CNV as siRNA inhibition is abolished in TLR3-deficient mice (Kleinman et al., 2008). Moreover, dsRNA causes RPE cell death that is mediated by TLR3 and genetic variant in the TLR3 412Phe confers protection against geographic atrophy (Yang et al., 2008). These observations reveal a role of TLR3 in AMD development.

#### **3.1.2 C-type lectin receptors (CLRs)**

32 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

(Kamei et al., 2007). Collectively, these observations suggest a causal role of oxLDL accumulation in AMD pathogenesis although the precise mechanisms are not well defined

A2E, a byproduct of the visual cycle, is formed through the condensation of two molecules of all-trans-retinal with one molecule of phosphatidylethanolamine in the POS upon photoisomerization of 11-*cis* retinal (Mata et al., 2000). A2E accumulates as lipofuscin in RPE cells with ageing since it is resistant to enzymatic degradation. After light, in particular blue light irradiation, A2E is oxidized, initially by addition of light-excited singlet oxygen, and oxidized A2E can then generate free radicals such as superoxide anion and hydroxyl radical, or discompose to reactive dicarbonyls like methylglyoxal, triggering free radical chain reaction (Wu et al., 2010). Light exposure causes death of A2E-loaded RPE cells in other hand A2E-free RPE cells are not (Schutt et al., 2000). Moreover, photooxidation products of A2E have been shown to activate complement system in RPE cells (Zhou et al., 2006), suggesting that sterile inflammation is involved in A2E cytotoxicity. However, a causal role

How does the innate immune system distinguish between dead and live cells? The crucial event upon necrotic cell death is the release of intracellular DAMPs and generation of extracellular DAMPs which can be sensed by the receptors on innate immune cells. This section discusses the recent progress in the recognition of DAMPs by pattern recognition receptors and DAMP

Pattern recognition receptors (PRRs) recognize conserved structural moieties called PAMPs found in microorganisms and in turn induce cytokine production, which is important in inflammatory and antimicrobial responses. Up to date, five classes of PRRs have been identified. They are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), the retinoic acidinducible gene (RIG)-I-like receptors (RLRs) and absence in melanoma 2 (AIM2). The membrane-bound TLRs and CLRs surveillance the extracellular milieu, whereas RLRs, NLRs and AIM2-like receptors have emerged as pivotal sensors of infection and stress in intracellular compartments (Meylan et al., 2006). Among them, TLRs, CLRs, NLRs and

TLRs have been reported to be activated by intracellular DAMPs including HSPs, S100 proteins, uric acid and HMGB1, as well as extracellular DAMPs such as hyaluronan, heparan sulphate and proteoglycans (Kono & Rock, 2008). RPE cells make up the first line of defense against pathogens by expressing almost all TLR iso-forms except TLR-8 (Kumar et al., 2004). Activation of TLR3 and TLR9 leads to the production of inflammatory mediators including cytokines and adhesion molecules in cultured RPE cells (Ebihara et al., 2007). TLR3 detects mRNA (Kariko et al., 2004) and dsRNA (Dogusan et al., 2008) released from

of A2E accumulation in AMD development remains to be confirmed.

receptors that are not typically associated with microbial recognition (Figure 1).

AIM2 participate in DAMP-dependent inflammatory responses.

**2.3.4 Bis-retinoid pyridinium A2E** 

**3. DAMP receptors** 

**3.1 Pattern recognition receptors** 

**3.1.1 Toll-like receptors (TLRs)** 

CLRs including DEC205, Mincle, CLEC9A and DC-SIGN are a family of surface receptors known to recognize carbohydrate moieties on viruses, bacteria and fungi (Cambi & Figdor, 2009). Stimulation of CLRs leads to activation of signaling pathways that elevate cytokine production. Although their ligands are poorly defined, Mincle (also known as CLEC4E) and CLEC9A can sense necrotic cell death (Cambi & Figdor, 2009). Mincle recognizes SAP130 from necrotic cells and triggers intracellular signaling via the associated FcRγ adaptor, leading to the production of inflammatory cytokines (Yamasaki et al., 2008). CLEC9A, a Sykcoupled CLR, can recognize necrotic cells and present dead cell-associated antigens to CD8+ T cells (Sancho et al., 2009). Similar to Mincle, the capability of CLEC9A to recognize necrotic cells makes it a potential receptor that is important for sterile inflammatory responses even though its ligands are unidentified.

#### **3.1.3 NOD-like receptors (NLRs)**

NLRs, consisting of the three subfamilies with 14 NALPs, 6 NODs and 2 IPAF/NAIP, are cytosolic PRRs that sense microbial invasion, eliciting an inflammatory response to alert the system to the presence of danger, mainly by assembling inflammasomes that activate caspase-1 for processing immature IL-1β to mature IL-1β (Martinon et al., 2009). NLRs contain a central nucleotide-binding oligomerization domain (NACHT), an N-terminal effector domain (pyrin domain, caspase-recruitment domain or BIR domain) and C-terminal leucine-rich repeats (LRRs). Among the NLRs identified so far, the NOD-, LRR-, and pyrin domain-containing 3 (NLRP3, also termed as NALP3) has been shown to be capable of detecting endogenous danger signals (*See* discussion in NLRP3 inflammasome).

#### **3.1.4 Absence in melanoma 2 (AIM2)**

AIM2 is a cytosolic protein containing a C-terminal HIN200 and an N-terminal PYD domain, which is identified to recognize dsDNA derived from virus and bacterial, triggering anti-virus responses (Burckstummer et al., 2009). AIM2 can also be activated by the transfection of synthetic dsDNA (Fernandes-Alnemri et al., 2009), highlighting that the innate response to DNA is regulated by the localization of DNA in concert with the innate receptors rather than the source of DNA. Under physiological conditions, self-DNA is localized in nuclei and mitochondria. However, injured or dying cells can release mitochondrial and genomic DNA into the cytosol where AIM2 resides. Released genomic and mitochondrial DNA have been demonstrated to cause inflammatory responses (Kawashima et al., 2011; Zhang et al., 2010). Whether there is a pathogenic role for AIM2 in sterile inflammation-related diseases remains unclear.

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 35

(Picard et al., 2010) as well as photoreceptor degeneration and choroidal involution (Houssier et al., 2008). Genetic analysis has identified that two common variants, rs3211883 and rs3173798 which do not reside in the coding sequence of *CD36* gene, are associated with

In response to necrotic cell death, the innate and adaptive immune systems respond with inflammation to contain and remove the offending agents. The mechanisms by which DAMPs trigger inflammatory responses are still not fully understood, however, the outcome of sterile inflammation to structurally diverse DAMPs is quite similar. Discussed here are activation of the NF-κB/AP-1 signaling and the NLRP3/AIM2 inflammasomes for

**4.1 Generation of inflammatory mediators by activating cell surface DAMP receptors**  Production of biologically active inflammatory mediators including cytokines and adhesion molecules during sterile injury-associated cell death is an important mechanism to alert the immune system of tissue damage and to initiate the healing response. Although the mechanistic details of these DAMP receptor signaling are not fully revealed yet, accumulating evidence shows that these DAMP receptors activate transcriptional factor NF-κB and AP-1, driving expression of inflammatory mediators required for initiating and

neovascular AMD in a Japanese population (Kondo et al., 2009).

**4. Mechanisms of sterile inflammation** 

generating inflammatory mediators.

promoting sterile inflammation (Figure 2).

Fig. 2. Overview of cell surface DAMP receptor signaling pathways.

#### **3.2 Non-PRR DAMP receptors**

DAMPs can also be recognized by non-PRRs, named as DAMP receptors here. Receptor for AGEs, purinergic P2X7 receptor and scavenger receptor CD36 are currently three relatively appreciated DAMP receptors.

#### **3.2.1 Receptor for AGEs (RAGE)**

RAGE is a transmembrane receptor that belongs to the immunoglobulin super-family of cell surface molecules that are constitutively expressed at very low levels in numerous cells, including Muller cells, photoreceptor cells, RPE cells, and vascular endothelial cells (Barile & Schmidt, 2007; Howes et al., 2004). It recognizes AGEs, HMGB1, amyloid-β (Bucciarelli et al., 2002) and the S100 family members (Hofmann et al., 1999). Activation of RAGE by its ligands results in the upregulation of several inflammatory signaling pathways, including NF-κB, phosphoinositide 3-kinase and MAPK signalling pathways, thereby producing inflammatory cytokines (Hofmann et al., 1999). Cellular expression of RAGE increases upon ligand binding, thus amplifying cellular activation. The levels of RAGE *in vivo* are correlated with drusen formation and early development of AMD (Howes et al., 2004). Transgenic expression of RAGE augmented blood-retinal barrier breakdown and leukostasis, accompanied by increased expression of VEGF and ICAM-1 in the retina in a murine diabetic model (Kaji et al., 2007), which were significantly inhibited by systemic administration of a soluble form of RAGE.

#### **3.2.2 Purinergic P2X7 receptor (P2X7R)**

The P2X7R belongs to the P2X receptor subfamily of P2 receptors (receptors for extracellular nucleotides) and is an ATP-gated cation channel that is widely expressed in cells of the immune system (Di Virgilio, 2007). Activation of P2X7R causes rapid efflux of K+ with accompanied influx of Ca2+ and Na+. ATP is the preferred agonist for P2X receptor subfamily, however, higher concentration of extracellular ATP is required for P2X7R activation than that required for the other P2X receptors (Surprenant et al., 1996). The known DAMPs that activate P2X7R are ATP and uric acid (Riteau et al., 2010). Knockout of P2X7R, or blockade of with antagonist inhibits ATP-dependent lung inflammation (Riteau et al., 2010) or hyperalgesia (Teixeira et al., 2010). Activation of P2X7R by ATP and synthetic ligand leads to RPE cell death *in vitro*, which is inhibited by P2X7R antagonist (Yang et al., 2010). Whether ATP- P2X7R is involved in RPE atrophy *in vivo* is unknown.

#### **3.2.3 Scavenger receptor CD36**

CD36 is a cell surface scavenger receptor expressed on RPE cells, which recognizes oxidized POS and facilitates their uptake by RPE cells (Sun et al., 2006). RPE cells can internalize LDL and oxLDL in large quantities *in vitro* and *in vivo* (Gordiyenko et al., 2004). Treatment with oxLDL induces transcriptional alterations in genes related to lipid metabolism, oxidative stress, inflammation and apoptosis in RPE cells (Yamada et al., 2008). Failure in oxLDL clearance further recruits macrophages via cell surface scavenger receptors to the sites where oxLDL accumulates, amplifying inflammatory responses via producing inflammatory cytokines (Kamei et al., 2007). Therefore, efficient recycle of shed POS and clearance of oxLDL are essential for eye health since CD36 deficiency in mice resulted in age-associated accumulation of oxLDL and sub-retinal Bruch's membrane thickening (Picard et al., 2010) as well as photoreceptor degeneration and choroidal involution (Houssier et al., 2008). Genetic analysis has identified that two common variants, rs3211883 and rs3173798 which do not reside in the coding sequence of *CD36* gene, are associated with neovascular AMD in a Japanese population (Kondo et al., 2009).

#### **4. Mechanisms of sterile inflammation**

34 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

DAMPs can also be recognized by non-PRRs, named as DAMP receptors here. Receptor for AGEs, purinergic P2X7 receptor and scavenger receptor CD36 are currently three relatively

RAGE is a transmembrane receptor that belongs to the immunoglobulin super-family of cell surface molecules that are constitutively expressed at very low levels in numerous cells, including Muller cells, photoreceptor cells, RPE cells, and vascular endothelial cells (Barile & Schmidt, 2007; Howes et al., 2004). It recognizes AGEs, HMGB1, amyloid-β (Bucciarelli et al., 2002) and the S100 family members (Hofmann et al., 1999). Activation of RAGE by its ligands results in the upregulation of several inflammatory signaling pathways, including NF-κB, phosphoinositide 3-kinase and MAPK signalling pathways, thereby producing inflammatory cytokines (Hofmann et al., 1999). Cellular expression of RAGE increases upon ligand binding, thus amplifying cellular activation. The levels of RAGE *in vivo* are correlated with drusen formation and early development of AMD (Howes et al., 2004). Transgenic expression of RAGE augmented blood-retinal barrier breakdown and leukostasis, accompanied by increased expression of VEGF and ICAM-1 in the retina in a murine diabetic model (Kaji et al., 2007), which were significantly inhibited by systemic administration of a soluble form of RAGE.

The P2X7R belongs to the P2X receptor subfamily of P2 receptors (receptors for extracellular nucleotides) and is an ATP-gated cation channel that is widely expressed in cells of the immune system (Di Virgilio, 2007). Activation of P2X7R causes rapid efflux of K+ with accompanied influx of Ca2+ and Na+. ATP is the preferred agonist for P2X receptor subfamily, however, higher concentration of extracellular ATP is required for P2X7R activation than that required for the other P2X receptors (Surprenant et al., 1996). The known DAMPs that activate P2X7R are ATP and uric acid (Riteau et al., 2010). Knockout of P2X7R, or blockade of with antagonist inhibits ATP-dependent lung inflammation (Riteau et al., 2010) or hyperalgesia (Teixeira et al., 2010). Activation of P2X7R by ATP and synthetic ligand leads to RPE cell death *in vitro*, which is inhibited by P2X7R antagonist (Yang et al.,

CD36 is a cell surface scavenger receptor expressed on RPE cells, which recognizes oxidized POS and facilitates their uptake by RPE cells (Sun et al., 2006). RPE cells can internalize LDL and oxLDL in large quantities *in vitro* and *in vivo* (Gordiyenko et al., 2004). Treatment with oxLDL induces transcriptional alterations in genes related to lipid metabolism, oxidative stress, inflammation and apoptosis in RPE cells (Yamada et al., 2008). Failure in oxLDL clearance further recruits macrophages via cell surface scavenger receptors to the sites where oxLDL accumulates, amplifying inflammatory responses via producing inflammatory cytokines (Kamei et al., 2007). Therefore, efficient recycle of shed POS and clearance of oxLDL are essential for eye health since CD36 deficiency in mice resulted in age-associated accumulation of oxLDL and sub-retinal Bruch's membrane thickening

2010). Whether ATP- P2X7R is involved in RPE atrophy *in vivo* is unknown.

**3.2 Non-PRR DAMP receptors** 

appreciated DAMP receptors.

**3.2.1 Receptor for AGEs (RAGE)** 

**3.2.2 Purinergic P2X7 receptor (P2X7R)** 

**3.2.3 Scavenger receptor CD36** 

In response to necrotic cell death, the innate and adaptive immune systems respond with inflammation to contain and remove the offending agents. The mechanisms by which DAMPs trigger inflammatory responses are still not fully understood, however, the outcome of sterile inflammation to structurally diverse DAMPs is quite similar. Discussed here are activation of the NF-κB/AP-1 signaling and the NLRP3/AIM2 inflammasomes for generating inflammatory mediators.

#### **4.1 Generation of inflammatory mediators by activating cell surface DAMP receptors**

Production of biologically active inflammatory mediators including cytokines and adhesion molecules during sterile injury-associated cell death is an important mechanism to alert the immune system of tissue damage and to initiate the healing response. Although the mechanistic details of these DAMP receptor signaling are not fully revealed yet, accumulating evidence shows that these DAMP receptors activate transcriptional factor NF-κB and AP-1, driving expression of inflammatory mediators required for initiating and promoting sterile inflammation (Figure 2).

Fig. 2. Overview of cell surface DAMP receptor signaling pathways.

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 37

Fig. 3. Intracellular DAMP receptors that sense upcoming danger.

kappa B.

**4.2.1 ROS generation** 

The nucleotide-binding oligomerization domain (NOD)-like receptor NLRP3 (NOD-, LRRand pyrin domain-containing 3) and the PYHIN (pyrin and HIN200 domain-containing) family protein AIM2 (absent in melanoma 2) are two PRRs that survilliance intracelluar DAMPs. NLRP3 can be activated by lowering intracellular potassium concentration, lysosomal damage-dependent activation of cathepsin B or generation of reactive oxygen species (ROS) during cellular stress or necrosis. On the other hand, intracellular DNA from damaged mitochondria or genomic DNA fragments activates AIM2 via binding to its HIN200 domain. Activated NLRP3 and AIM2 provide binding sites for the adaptor ASC (apoptosis-related speck-like protein) via homotypic pyrin domain (PYD) interactions. Clustered ASC then recruits pro-caspase-1 through caspase recruitment domain (CARD)- CARD interactions for assembling NLRP3 or AIM2 inflammasome. The NLRP3 and AIM2 inflammasomes activate caspase-1 which subsequently processes pro-cytokines interleukin-1β (IL-1β) and IL-18 into their active forms. AP-1, activator protein-1. NF-κB, nuclear factor

The detrimental effect of ROS during sterile inflammation depends on the balance between ROS producers and ROS detoxification by antioxidants. ROS are the common integrator across silica, MSU (Dostert et al., 2008) and ATP (Cruz et al., 2007) that activate the NLRP3 inflammasome. ROS removal by *N*-acetyl-L-cysteine, DPI-inhibition of NADPH oxidase and p22phox knockdown resulted in impairment of caspase 1 activation and IL-1β production by these stimuli (Cruz et al., 2007; Dostert et al., 2008). The causal role of ROS in NLRP3 activation is further confirmed by Zhou *et al*. (Zhou et al., 2011) and Nakahira *et al*. (Nakahira et al., 2011),

The presentation of DAMP molecules to host cells triggers the sequential activation of signaling cascades that activate transcriptional factors NF-κB and AP-1, leading to production of inflammatory mediators. AP-1, activator protein-1. Bcl10, B cell lymphoma 10. CARD9, caspase recruitment domain-containing protein-9. ITAM, immunoreceptor tyrosine-based activation motif. MyD88, myeloid differentiation primary-response gene 88. IKK, inhibitor of kappa B kinase. IRAK, IL-1 receptor-associated kinase. MAPK, mitogen-activated kinase. NFκB, nuclear factor kappa B. SFK, src-family kinases. Syk, spleen tyrosine kinase. TAK1, transforming growth factor β–activated kinase 1. TRAF6, TNF receptor-associated factor 6. TRIF, TIR-domain-containing adapter-inducing interferon-β.

The signals relayed from activated TLR2, TLR3, TLR4 and IL-1R convene at TNF receptorassociated factor 6 (TRAF6) that further activates NF-κB through TAK1-IKK or activates AP-1 through TAK1-MAPK (Sloane et al., 2010). On the other hand, CLRs Mincle and CLEC9A couple with FcRγ adaptor and activate transcriptional factor NF-κB through Syk kinasecaspase recruitment domain-containing protein-9 (CARD9) pathway (Drummond et al., 2011). Activated P2X7R (Skaper et al., 2010) and CD36 (Stuart et al., 2007) first promote activation of src-family kinases that trigger NF-κB- and AP-1-dependent transcription via small G-protein-MAPK pathways. Although the mechanistic details of RAGE signaling and the importance of its various ligands in disease pathology continue to be areas of investigation, activated RAGE by its various ligands also leads to activation of transcription factors NF-κB and AP-1 by ras-MAPK signaling (Glenn & Stitt, 2009). Activated NF-κB and AP-1 finally drive expression of inflammatory mediators, promoting sterile inflammation. Among the inflammatory cytokines synthesized, IL-1β and IL-18 are produced and stored in cytosol as inactive precursors that are then converted into biological active forms by the activated NLRP3 and AIM2 inflammasomes through caspase 1-dependent proteolytic maturation, which will be discussed below.

#### **4.2 Production of active IL-1β by NLRP3 inflammasome**

IL-1β is a potent pro-inflammatory cytokine that is important in sterile inflammation by induction of inflammatory mediators (Gabay et al., 2010). IL-1β and IL-18 are synthesized and stored in the cytosol as inactive precursors. The processing and secretion of active IL-1β and IL-18 by inflammatory cells depend largely on the inflammasomes, of which the hallmark is the activation of caspase 1 responsible for processing immature IL-1β and IL-18 into their biologically active forms (Martinon et al., 2009). Among several inflammasomes described up to date, NLRP3 (also named as NALP3) inflammasome has been demonstrated to sense DAMP molecules in sterile inflammatory responses. Understanding how NLRP3 senses diverse sterile stimuli is important for understanding the pathogenesis of possibly many sterile inflammatory disorders and for identifying potential therapeutic targets. NLRP3 inflammasome consists of NLRP3, adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 (Martinon et al., 2009). NLRP3 detects the intracellular ligands by its C-terminal LRR domain, triggering oligomerization by NACHT domain interaction in an ATP-dependent manner followed by caspase-1 recruitment and activation by autocleavage. NLRP3 does not sense structurally diverse stimuli individually, but rather senses a common downstream event mainly through three pathways: ROS generation, lysosome rupture and lowering intracellular potassium (Figure 3).

Fig. 3. Intracellular DAMP receptors that sense upcoming danger.

The nucleotide-binding oligomerization domain (NOD)-like receptor NLRP3 (NOD-, LRRand pyrin domain-containing 3) and the PYHIN (pyrin and HIN200 domain-containing) family protein AIM2 (absent in melanoma 2) are two PRRs that survilliance intracelluar DAMPs. NLRP3 can be activated by lowering intracellular potassium concentration, lysosomal damage-dependent activation of cathepsin B or generation of reactive oxygen species (ROS) during cellular stress or necrosis. On the other hand, intracellular DNA from damaged mitochondria or genomic DNA fragments activates AIM2 via binding to its HIN200 domain. Activated NLRP3 and AIM2 provide binding sites for the adaptor ASC (apoptosis-related speck-like protein) via homotypic pyrin domain (PYD) interactions. Clustered ASC then recruits pro-caspase-1 through caspase recruitment domain (CARD)- CARD interactions for assembling NLRP3 or AIM2 inflammasome. The NLRP3 and AIM2 inflammasomes activate caspase-1 which subsequently processes pro-cytokines interleukin-1β (IL-1β) and IL-18 into their active forms. AP-1, activator protein-1. NF-κB, nuclear factor kappa B.

#### **4.2.1 ROS generation**

36 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

The presentation of DAMP molecules to host cells triggers the sequential activation of signaling cascades that activate transcriptional factors NF-κB and AP-1, leading to production of inflammatory mediators. AP-1, activator protein-1. Bcl10, B cell lymphoma 10. CARD9, caspase recruitment domain-containing protein-9. ITAM, immunoreceptor tyrosine-based activation motif. MyD88, myeloid differentiation primary-response gene 88. IKK, inhibitor of kappa B kinase. IRAK, IL-1 receptor-associated kinase. MAPK, mitogen-activated kinase. NFκB, nuclear factor kappa B. SFK, src-family kinases. Syk, spleen tyrosine kinase. TAK1, transforming growth factor β–activated kinase 1. TRAF6, TNF receptor-associated factor 6.

The signals relayed from activated TLR2, TLR3, TLR4 and IL-1R convene at TNF receptorassociated factor 6 (TRAF6) that further activates NF-κB through TAK1-IKK or activates AP-1 through TAK1-MAPK (Sloane et al., 2010). On the other hand, CLRs Mincle and CLEC9A couple with FcRγ adaptor and activate transcriptional factor NF-κB through Syk kinasecaspase recruitment domain-containing protein-9 (CARD9) pathway (Drummond et al., 2011). Activated P2X7R (Skaper et al., 2010) and CD36 (Stuart et al., 2007) first promote activation of src-family kinases that trigger NF-κB- and AP-1-dependent transcription via small G-protein-MAPK pathways. Although the mechanistic details of RAGE signaling and the importance of its various ligands in disease pathology continue to be areas of investigation, activated RAGE by its various ligands also leads to activation of transcription factors NF-κB and AP-1 by ras-MAPK signaling (Glenn & Stitt, 2009). Activated NF-κB and AP-1 finally drive expression of inflammatory mediators, promoting sterile inflammation. Among the inflammatory cytokines synthesized, IL-1β and IL-18 are produced and stored in cytosol as inactive precursors that are then converted into biological active forms by the activated NLRP3 and AIM2 inflammasomes through caspase 1-dependent proteolytic

IL-1β is a potent pro-inflammatory cytokine that is important in sterile inflammation by induction of inflammatory mediators (Gabay et al., 2010). IL-1β and IL-18 are synthesized and stored in the cytosol as inactive precursors. The processing and secretion of active IL-1β and IL-18 by inflammatory cells depend largely on the inflammasomes, of which the hallmark is the activation of caspase 1 responsible for processing immature IL-1β and IL-18 into their biologically active forms (Martinon et al., 2009). Among several inflammasomes described up to date, NLRP3 (also named as NALP3) inflammasome has been demonstrated to sense DAMP molecules in sterile inflammatory responses. Understanding how NLRP3 senses diverse sterile stimuli is important for understanding the pathogenesis of possibly many sterile inflammatory disorders and for identifying potential therapeutic targets. NLRP3 inflammasome consists of NLRP3, adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 (Martinon et al., 2009). NLRP3 detects the intracellular ligands by its C-terminal LRR domain, triggering oligomerization by NACHT domain interaction in an ATP-dependent manner followed by caspase-1 recruitment and activation by autocleavage. NLRP3 does not sense structurally diverse stimuli individually, but rather senses a common downstream event mainly through three pathways: ROS generation, lysosome rupture

TRIF, TIR-domain-containing adapter-inducing interferon-β.

maturation, which will be discussed below.

and lowering intracellular potassium (Figure 3).

**4.2 Production of active IL-1β by NLRP3 inflammasome** 

The detrimental effect of ROS during sterile inflammation depends on the balance between ROS producers and ROS detoxification by antioxidants. ROS are the common integrator across silica, MSU (Dostert et al., 2008) and ATP (Cruz et al., 2007) that activate the NLRP3 inflammasome. ROS removal by *N*-acetyl-L-cysteine, DPI-inhibition of NADPH oxidase and p22phox knockdown resulted in impairment of caspase 1 activation and IL-1β production by these stimuli (Cruz et al., 2007; Dostert et al., 2008). The causal role of ROS in NLRP3 activation is further confirmed by Zhou *et al*. (Zhou et al., 2011) and Nakahira *et al*. (Nakahira et al., 2011),

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 39

inhibiting DAMP-initiated inflammatory signaling could potentially delay the onset or progression of this disease. A number of promising anti-DAMP agents are currently under

As strong association of complement alternative pathway activation with higher AMD risk has been documented, suppressing complement cascade in the retina would be expected to delay or reverse the onset of AMD. Inhibition of complement alternative pathway can be achieved via targeting factor B and factor D which participate in the amplification of the complement alternative pathway, or inhibiting targets downstream the point of conversion for all three complement pathways. TA-106 and TNX-234, anti-Factor B and anti-Factor D antibodies, are in preclinical testing and Phase I/II trial for AMD by Alexion and Genentech, respectively. JPE-1375 is a small molecule peptidomimetic antagonist targeting the C5a receptor (Ricklin & Lambris, 2007) that is in preclinical evaluation for AMD. ARC-1905, a 39 mer oligonucleotide anti-C5 aptamer is in Phase 1 for AMD by Ophthotech. POT-4 is a cyclic peptide capable of binding to human C3, resulting in broad and potent complement activation inhibition (Ricklin & Lambris, 2007). POT-4 is the first complement inhibitor that has entered into a Phase I clinical trial for AMD by Potentia and is now under development

atrophy Genentech Phase I/II Unknown

atrophy Jerini Pre-clinical

ARC-1905 AMD Ophthotech Phase I Mar. 2011

POT-4 AMD Alcon Phase 1 Feb. 2010

Canakinumab Wet AMD Novartis Phase 1 Dec. 2007

Massachusetts

Table 1. Developing anti-DAMP therapies for age-related macular degeneration

dementia Pfizer Phase II Dec. 2010

Given the crucial role for IL-1α and IL-1β in sterile inflammatory responses, blocking IL-1 receptor is expected to benefit patients with sterile inflammatory disorders. Some promising results have been obtained with the application of a recombinant IL-1 receptor antagonist,

development

testing

Eye Infirmary Phase I/II Under

Estimated completion day

testing Unknown Anti-factor

ongoing

ongoing

completed

completed

development

completed

Under development Mechanism

B antibody

Anti-factor D antibody

C5aR antagonist

C3 inhibitor

IL-1 receptor antagonist

RAGE inhibitor

Anti-C5 aptamer

Anti-IL1β antibody

of activation

development for AMD therapy, some of them are still in preclinical testing (Table 1).

**5.1 Complement alternative pathway inhibitors** 

Compound Indications Company State of

TA-106 AMD & asthma Alexion Pre-clinical

by Alcon (Table 1).

TNX-234 Geographic

JPE-1375 Geographic

Anakinra Corneal

PF-04494700 Alzheimer's

**5.2 IL-1 pathway inhibitor** 

neovascularization

identifying that mitochondrial ROS derived from complex I and III are responsible for NLRP3 activation. Although it is undefined how NLRP3 senses ROS, redox imbalance may be one of the unifying mechanisms by which NLRP3 senses its various activators.

#### **4.2.2 Lowering intracellular potassium**

Potassium efflux has been suggested to be an essential upstream signal of NLRP3 activation. Blocking potassium efflux in cultured media abrogates NLRP3 activation by asbestos, MSU and ATP (Dostert et al., 2008). Extracellular ATP is known to open an ATP-gated cation channel that causes potassium efflux via binding to purinergic receptor P2X7R. Antibiotics such as neomycin and gramicidin stimulate NLRP3 inflammasome-dependent secretion of IL-1β, depending on potassium efflux but independent of P2X7R (Allam et al., 2011). Passive water influx due to sodium overload, which only dilutes cytoplasmic potassium ions, is able to activate NLRP3 inflammasome (Schorn et al., 2011). Thus, NLRP3 senses intracellular K+ depletion, regardless whether it is due to the activation of specific ion channels or a nonselective increase in ion permeability as a result of cell injury.

#### **4.2.3 Lysosomal rupture**

Sterile crystalline and particulate activators of the NLRP3 inflammasome such as urate and cholesterol crystals cause lysosomal destabilization and cathepsin B release, which can be detected by NLRP3. Activation of NLRP3 inflammasome by amyloid-β (Halle et al., 2008) and silica and MSU crystals (Hornung et al., 2008) requires their internalization through endocytosis and lysosomal cathepsin B activation as inhibition of endocytosis or cathepsin B impairs NLRP3-dependent IL-1β production. The same is true to cholesterol crystals that activate NLRP3 inflammasome in a cathepsin B-dependent manner (Rajamaki et al., 2010). Lysosomal damage can occur during cellular injury and necrosis, therefore, lysosomal destabilization may be one of the converging points of divergent danger signals sensed by NRLP3. However, whether there is a common mechanism by which numerous heterogeneous stimuli converge on NLRP3 downstream of ROS generation, potassium efflux and lysosomal rupture, remains to be determined.

#### **4.3 Production of active IL-1β by AIM2 inflammasome**

In addition to the NLRP3 inflammasome, the AIM2 inflammasome is the second one indentified so far to be involved in sterile inflammation. In contrast to NLRP3, AIM2 has a highly restricted spectrum of activating stimuli, currently known being involved in sensing cytosolic dsDNA regardless of the DNA source. The HIN200 domain in its C terminus interacts directly with dsDNA and triggers recruitment and activation of caspase-1 in an ASC-dependent manner via its N-terminal pyrin domain, thereby processing IL-1β and IL-18 into their active forms (Hornung et al., 2009). Mitochondrial DNA (Zhang et al., 2010) and genomic DNA fragments (Kawashima et al., 2011) released from cellular injury can trigger inflammatory responses through activation of the AIM2 inflammasome in addition to TLR9.

#### **5. Therapeutic potentials of anti-DAMP for AMD**

Cell injury by chronic sterile inflammation leads to progressive loss of cell function and thus contributes to the development of AMD. Protecting retinal cells by neutralizing DAMPs and inhibiting DAMP-initiated inflammatory signaling could potentially delay the onset or progression of this disease. A number of promising anti-DAMP agents are currently under development for AMD therapy, some of them are still in preclinical testing (Table 1).

#### **5.1 Complement alternative pathway inhibitors**

38 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

identifying that mitochondrial ROS derived from complex I and III are responsible for NLRP3 activation. Although it is undefined how NLRP3 senses ROS, redox imbalance may be one of

Potassium efflux has been suggested to be an essential upstream signal of NLRP3 activation. Blocking potassium efflux in cultured media abrogates NLRP3 activation by asbestos, MSU and ATP (Dostert et al., 2008). Extracellular ATP is known to open an ATP-gated cation channel that causes potassium efflux via binding to purinergic receptor P2X7R. Antibiotics such as neomycin and gramicidin stimulate NLRP3 inflammasome-dependent secretion of IL-1β, depending on potassium efflux but independent of P2X7R (Allam et al., 2011). Passive water influx due to sodium overload, which only dilutes cytoplasmic potassium ions, is able to activate NLRP3 inflammasome (Schorn et al., 2011). Thus, NLRP3 senses intracellular K+ depletion, regardless whether it is due to the activation of specific ion channels or a non-

Sterile crystalline and particulate activators of the NLRP3 inflammasome such as urate and cholesterol crystals cause lysosomal destabilization and cathepsin B release, which can be detected by NLRP3. Activation of NLRP3 inflammasome by amyloid-β (Halle et al., 2008) and silica and MSU crystals (Hornung et al., 2008) requires their internalization through endocytosis and lysosomal cathepsin B activation as inhibition of endocytosis or cathepsin B impairs NLRP3-dependent IL-1β production. The same is true to cholesterol crystals that activate NLRP3 inflammasome in a cathepsin B-dependent manner (Rajamaki et al., 2010). Lysosomal damage can occur during cellular injury and necrosis, therefore, lysosomal destabilization may be one of the converging points of divergent danger signals sensed by NRLP3. However, whether there is a common mechanism by which numerous heterogeneous stimuli converge on NLRP3 downstream of ROS generation, potassium

In addition to the NLRP3 inflammasome, the AIM2 inflammasome is the second one indentified so far to be involved in sterile inflammation. In contrast to NLRP3, AIM2 has a highly restricted spectrum of activating stimuli, currently known being involved in sensing cytosolic dsDNA regardless of the DNA source. The HIN200 domain in its C terminus interacts directly with dsDNA and triggers recruitment and activation of caspase-1 in an ASC-dependent manner via its N-terminal pyrin domain, thereby processing IL-1β and IL-18 into their active forms (Hornung et al., 2009). Mitochondrial DNA (Zhang et al., 2010) and genomic DNA fragments (Kawashima et al., 2011) released from cellular injury can trigger inflammatory responses through activation of the AIM2 inflammasome in addition to TLR9.

Cell injury by chronic sterile inflammation leads to progressive loss of cell function and thus contributes to the development of AMD. Protecting retinal cells by neutralizing DAMPs and

the unifying mechanisms by which NLRP3 senses its various activators.

selective increase in ion permeability as a result of cell injury.

efflux and lysosomal rupture, remains to be determined.

**4.3 Production of active IL-1β by AIM2 inflammasome** 

**5. Therapeutic potentials of anti-DAMP for AMD** 

**4.2.2 Lowering intracellular potassium** 

**4.2.3 Lysosomal rupture** 

As strong association of complement alternative pathway activation with higher AMD risk has been documented, suppressing complement cascade in the retina would be expected to delay or reverse the onset of AMD. Inhibition of complement alternative pathway can be achieved via targeting factor B and factor D which participate in the amplification of the complement alternative pathway, or inhibiting targets downstream the point of conversion for all three complement pathways. TA-106 and TNX-234, anti-Factor B and anti-Factor D antibodies, are in preclinical testing and Phase I/II trial for AMD by Alexion and Genentech, respectively. JPE-1375 is a small molecule peptidomimetic antagonist targeting the C5a receptor (Ricklin & Lambris, 2007) that is in preclinical evaluation for AMD. ARC-1905, a 39 mer oligonucleotide anti-C5 aptamer is in Phase 1 for AMD by Ophthotech. POT-4 is a cyclic peptide capable of binding to human C3, resulting in broad and potent complement activation inhibition (Ricklin & Lambris, 2007). POT-4 is the first complement inhibitor that has entered into a Phase I clinical trial for AMD by Potentia and is now under development by Alcon (Table 1).


Table 1. Developing anti-DAMP therapies for age-related macular degeneration

#### **5.2 IL-1 pathway inhibitor**

Given the crucial role for IL-1α and IL-1β in sterile inflammatory responses, blocking IL-1 receptor is expected to benefit patients with sterile inflammatory disorders. Some promising results have been obtained with the application of a recombinant IL-1 receptor antagonist,

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration 41

Allam R, Darisipudi MN, Rupanagudi KV, Lichtnekert J, Tschopp J & Anders HJ (2011)

Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O, Erlandsson-Harris H, Janson

Arimura N, Ki-i Y, Hashiguchi T, Kawahara K, Biswas KK, Nakamura M, Sonoda Y,

Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC &

Babelova A, Moreth K, Tsalastra-Greul W, Zeng-Brouwers J, Eickelberg O, Young MF,

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anakinra. Anakinra is in clinic for the treatment of rheumatoid arthritis (Mertens & Singh, 2009). Phase I/II clinical trials are ongoing for the topical treatment of corneal neovascularization by Massachusetts Eye and Ear Infirmary. Intravitreal delivery of anakinra significantly inhibits experimental CNV in animal model (Olson et al., 2009). A benefit for patients with AMD is expecting with use of anakinra though no clinical trial is initiated. Neutralizing ligands for IL-1 receptor on the other hand would achieve similar outcomes. IL-1β antibody, canakinumab, has been approved for treating cryopyinassociated periodic syndromes. Canakinumab is under Phase I evaluation for choroidal neovascurilization by Norvartis.

#### **5.3 Anti-RAGE**

The levels of RAGE, which detects AGEs, S100 proteins, HMGB1 and amyloid-β, are very lower but dramatically increases on the Bruch's membrane around drusen and are associated with early development of AMD. Inhibiting RAGE receptor will block multiligand-triggered inflammatory response, thereby delaying or preventing the onset and progression of the disease. Pfizer is conducting a Phase II trial of RAGE inhibitor, PF-04494700, for treating Alzheimer's dementia, which share similar risk factors with AMD.

#### **5.4 Anti-TLR-3**

Activation of TLR3 by heterologous RNA from necrotic cells or *Alu* RNA causes RPE cell death and retinal degeneration. TLR3 knockout and TLR3 412Phe provents retinal degeneration in animals and confers protection against geographic atrophy in patients, respectively. TLR3 monoclonal antibody 23C8 is currently in Phase I trial for treatment of inflammation by Innate. No trial has been reported for AMD yet.

#### **6. Conclusion**

Cells or tissues incite sterile inflammatory responses to clear and repair the damage by sensing DAMPs derived from necrotic cells. Persistent inflammation causes cell dysfunction and subsequent retinal degeneration. Much progress has been made in identifying sterile inflammatory triggers and understanding the molecule mechanisms, which offers opportunities to design novel targets for delaying and preventing onset of retinal diseases including AMD. However, many questions remain to be answered. As cells contain many DAMPs, which DAMPs are the most important and whether their relative importance depends on cell types or pathophysiological conditions? How do different DAMPs initiate a common sterile inflammatory response and their downstream signaling pathways? Also unknown is the significance of DAMP-receptor interactions in sterile inflammation and disease pathogenesis since some DAMPs bind to several receptors and vice versa. Once the molecular mechanisms by which necrosis triggers sterile inflammation are elucidated and the relative importance of DAMPs is determined, one can manipulate the immune response to treat and manage sterile inflammation-associated diseases.

#### **7. References**

Alam MU, Harken JA, Knorn AM, Elford AR, Wigmore K, Ohashi PS & Millar DG (2009) Transgenic expression of Hsc70 in pancreatic islets enhances autoimmune diabetes in response to beta cell damage. J Immunol 183:5728-5737.

anakinra. Anakinra is in clinic for the treatment of rheumatoid arthritis (Mertens & Singh, 2009). Phase I/II clinical trials are ongoing for the topical treatment of corneal neovascularization by Massachusetts Eye and Ear Infirmary. Intravitreal delivery of anakinra significantly inhibits experimental CNV in animal model (Olson et al., 2009). A benefit for patients with AMD is expecting with use of anakinra though no clinical trial is initiated. Neutralizing ligands for IL-1 receptor on the other hand would achieve similar outcomes. IL-1β antibody, canakinumab, has been approved for treating cryopyinassociated periodic syndromes. Canakinumab is under Phase I evaluation for choroidal

The levels of RAGE, which detects AGEs, S100 proteins, HMGB1 and amyloid-β, are very lower but dramatically increases on the Bruch's membrane around drusen and are associated with early development of AMD. Inhibiting RAGE receptor will block multiligand-triggered inflammatory response, thereby delaying or preventing the onset and progression of the disease. Pfizer is conducting a Phase II trial of RAGE inhibitor, PF-04494700, for treating Alzheimer's dementia, which share similar risk factors with AMD.

Activation of TLR3 by heterologous RNA from necrotic cells or *Alu* RNA causes RPE cell death and retinal degeneration. TLR3 knockout and TLR3 412Phe provents retinal degeneration in animals and confers protection against geographic atrophy in patients, respectively. TLR3 monoclonal antibody 23C8 is currently in Phase I trial for treatment of

Cells or tissues incite sterile inflammatory responses to clear and repair the damage by sensing DAMPs derived from necrotic cells. Persistent inflammation causes cell dysfunction and subsequent retinal degeneration. Much progress has been made in identifying sterile inflammatory triggers and understanding the molecule mechanisms, which offers opportunities to design novel targets for delaying and preventing onset of retinal diseases including AMD. However, many questions remain to be answered. As cells contain many DAMPs, which DAMPs are the most important and whether their relative importance depends on cell types or pathophysiological conditions? How do different DAMPs initiate a common sterile inflammatory response and their downstream signaling pathways? Also unknown is the significance of DAMP-receptor interactions in sterile inflammation and disease pathogenesis since some DAMPs bind to several receptors and vice versa. Once the molecular mechanisms by which necrosis triggers sterile inflammation are elucidated and the relative importance of DAMPs is determined, one can manipulate the immune response

Alam MU, Harken JA, Knorn AM, Elford AR, Wigmore K, Ohashi PS & Millar DG (2009)

in response to beta cell damage. J Immunol 183:5728-5737.

Transgenic expression of Hsc70 in pancreatic islets enhances autoimmune diabetes

inflammation by Innate. No trial has been reported for AMD yet.

to treat and manage sterile inflammation-associated diseases.

neovascurilization by Norvartis.

**5.3 Anti-RAGE** 

**5.4 Anti-TLR-3** 

**6. Conclusion** 

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C, Caspers L, Boeynaems JM & Willermain F (2009) Extracellular nucleotides and interleukin-8 production by ARPE cells: potential role of danger signals in blood-

Quesniaux VF, Marchand-Adam S, Crestani B, Ryffel B & Couillin I (2010) Extracellular ATP is a danger signal activating P2X7 receptor in lung inflammation

cultured RPE cells is dependent on the formation of 7-ketocholesterol. Invest

of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis

Reis e Sousa C (2009) Identification of a dendritic cell receptor that couples sensing

MF, Mihalik D, Gotte M, Malle E, Schaefer RM & Grone HJ (2005) The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4

product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging.


Wu Y, Yanase E, Feng X, Siegel MM & Sparrow JR (2010) Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration. Proc Natl Acad Sci U S A 107:7275-7280.

**3** 

*Iowa City, Iowa* 

*USA* 

**Bruch's Membrane: The Critical** 

Robert F. Mullins and Elliott H. Sohn

*The University of Iowa Institute for Vision Research The Department of Ophthalmology and Visual Sciences The University of Iowa Carver College of Medicine* 

**Boundary in Macular Degeneration** 

In the early second century A.D., the Roman Empire had reached its zenith. The *pax romana* extended to all the lands touching the Mediterranean, counterclockwise from Northern Africa to Palestine, through Asia Minor, Gaul and Hispania, all the way to modern Wales and England. This last province of Britannia was visited by the emperor Hadrian, where he put on a show of force in response to a recent uprising. Most famously, during this visit Hadrian ordered the construction of a set of earthworks that now bears his name, stretching nearly eighty miles from Carlisle to Newcastle. Studded with fortifications, and manned with highly trained foreign legionnaires, this imposing structure stretched the width of the island and its ruins are impressive still today (Figure 1). While there is disagreement about the principal purpose of Hadrian's Wall—whether primarily as a symbolic marker of the northern extent of the Empire, a defensive fortification, or as a means of regulating commerce—the Historia Augusta states that the wall was constructed *qui barbaros* 

*Romanosque divideret;* to separate the Romans from the Barbarians(Magie, 1921).

safe barrier between civilization and chaos.

Whatever the principal motivation for its construction, Hadrian's wall did stand at the frontier between the Roman controlled territory and the Scots, Picts and other uncontrollable northern tribes of reputed violence. To the mind of the most Romanized Britons, who never met one of their northern neighbors, the wall must have represented the

Like the boundary between the civilized and barbarian world, a similar barrier stands at the threshold between the neural retina and the blood. The photoreceptor cells of the human retina face a dilemma. These cells are extremely energetic, consuming large quantities of oxygen. Elegant physiological studies by Linsenmeier and colleagues have shown that the oxygen tension from the RPE to the outer nuclear layer plunges over a distance of about 50µm, and that consumption at the level of the inner segment is dramatic(Birol, et al., 2007). Thus, the outer retina has a requirement for a large vascular supply. This requirement is met by the choriocapillaris, a unique vascular bed beneath the RPE. The endothelial cells (EC) of the choriocapillaris, like other EC, appear to use very little of the oxygen they deliver, instead likely relying largely on glycolytic metabolism. The choroid receives the vast

**1. Introduction** 


### **Bruch's Membrane: The Critical Boundary in Macular Degeneration**

Robert F. Mullins and Elliott H. Sohn

*The University of Iowa Institute for Vision Research The Department of Ophthalmology and Visual Sciences The University of Iowa Carver College of Medicine Iowa City, Iowa USA* 

#### **1. Introduction**

48 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Wu Y, Yanase E, Feng X, Siegel MM & Sparrow JR (2010) Structural characterization of

Yamada Y, Ishibashi K, Bhutto IA, Tian J, Lutty GA & Handa JT (2006) The expression of

Yamada Y, Tian J, Yang Y, Cutler RG, Wu T, Telljohann RS, Mattson MP & Handa JT (2008)

Yamasaki S, Ishikawa E, Sakuma M, Hara H, Ogata K & Saito T (2008) Mincle is an ITAMcoupled activating receptor that senses damaged cells. Nat Immunol 9:1179-1188. Yang D, Elner SG, Clark AJ, Hughes BA, Petty HR & Elner VM (2010) Activation of P2X

Yang Z, Stratton C, Francis PJ, Kleinman ME, Tan PL, Gibbs D, Tong Z, Chen H,

Yu AL, Lorenz RL, Haritoglou C, Kampik A & Welge-Lussen U (2009) Biological effects of

Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, Brohi K, Itagaki K & Hauser CJ

Zhou J, Jang YP, Kim SR & Sparrow JR (2006) Complement activation by photooxidation

Zhou R, Yazdi AS, Menu P & Tschopp J (2011) A role for mitochondria in NLRP3

Atrophy in Age-Related Macular Degeneration. N Engl J Med.

degeneration. Proc Natl Acad Sci U S A 107:7275-7280.

pigmented epithelial cells. J Neurochem 105:1187-1197.

in human maculas. Exp Eye Res 82:840-848.

Ophthalmol Vis Sci 52:1522-1530.

epithelial cells. Exp Eye Res 88:495-503.

Natl Acad Sci U S A 103:16182-16187.

inflammasome activation. Nature 469:221-225.

Nature 464:104-107.

bisretinoid A2E photocleavage products and implications for age-related macular

advanced glycation endproduct receptors in rpe cells associated with basal deposits

Oxidized low density lipoproteins induce a pathologic response by retinal

Receptors Induces Apoptosis in Human Retinal Pigment Epithelium. Invest

Constantine R, Yang X, Chen Y, Zeng J, Davey L, Ma X, Hau VS, Wang C, Harmon J, Buehler J, Pearson E, Patel S, Kaminoh Y, Watkins S, Luo L, Zabriskie NA, Bernstein PS, Cho W, Schwager A, Hinton DR, Klein ML, Hamon SC, Simmons E, Yu B, Campochiaro B, Sunness JS, Campochiaro P, Jorde L, Parmigiani G, Zack DJ, Katsanis N, Ambati J & Zhang K (2008) Toll-like Receptor 3 and Geographic

native and oxidized low-density lipoproteins in cultured human retinal pigment

(2010) Circulating mitochondrial DAMPs cause inflammatory responses to injury.

products of A2E, a lipofuscin constituent of the retinal pigment epithelium. Proc

In the early second century A.D., the Roman Empire had reached its zenith. The *pax romana* extended to all the lands touching the Mediterranean, counterclockwise from Northern Africa to Palestine, through Asia Minor, Gaul and Hispania, all the way to modern Wales and England. This last province of Britannia was visited by the emperor Hadrian, where he put on a show of force in response to a recent uprising. Most famously, during this visit Hadrian ordered the construction of a set of earthworks that now bears his name, stretching nearly eighty miles from Carlisle to Newcastle. Studded with fortifications, and manned with highly trained foreign legionnaires, this imposing structure stretched the width of the island and its ruins are impressive still today (Figure 1). While there is disagreement about the principal purpose of Hadrian's Wall—whether primarily as a symbolic marker of the northern extent of the Empire, a defensive fortification, or as a means of regulating commerce—the Historia Augusta states that the wall was constructed *qui barbaros Romanosque divideret;* to separate the Romans from the Barbarians(Magie, 1921).

Whatever the principal motivation for its construction, Hadrian's wall did stand at the frontier between the Roman controlled territory and the Scots, Picts and other uncontrollable northern tribes of reputed violence. To the mind of the most Romanized Britons, who never met one of their northern neighbors, the wall must have represented the safe barrier between civilization and chaos.

Like the boundary between the civilized and barbarian world, a similar barrier stands at the threshold between the neural retina and the blood. The photoreceptor cells of the human retina face a dilemma. These cells are extremely energetic, consuming large quantities of oxygen. Elegant physiological studies by Linsenmeier and colleagues have shown that the oxygen tension from the RPE to the outer nuclear layer plunges over a distance of about 50µm, and that consumption at the level of the inner segment is dramatic(Birol, et al., 2007). Thus, the outer retina has a requirement for a large vascular supply. This requirement is met by the choriocapillaris, a unique vascular bed beneath the RPE. The endothelial cells (EC) of the choriocapillaris, like other EC, appear to use very little of the oxygen they deliver, instead likely relying largely on glycolytic metabolism. The choroid receives the vast

Bruch's Membrane: The Critical Boundary in Macular Degeneration 51

**2. Clinical relevance of Bruch's membrane dysfunction: Age-related macular** 

The most common ocular condition of elderly humans with BrM dysfunction is age-related macular degeneration (AMD). This disease affects the center of the vision by damaging the macula, the critical portion of the retina responsible for fine visual acuity and daily activities such as reading, driving and recognizing faces. It is the most common cause of blindness in the elderly population in the western world(Klein, et al., 1992; Mitchell, et al., 1995; Vingerling, et al., 1995). Genetic variants conferring risk for developing AMD include mutations in complement factor H (*CFH*), complement component 2 (*C2*), *C3, CFB*, and the age-related maculopathy susceptibility 2/HtrA serine peptidase 1 (*ARMS2/HTRA1*)(Bergeron-Sawitzke, et al., 2009; Edwards, et al., 2005; Fritsche, et al., 2008; Hageman, et al., 2005; Haines, et al., 2005; Kanda, et al., 2007; Klein, et al., 2005; Maller, et al., 2007; Rivera, et al., 2005; Yates, et al., 2007); also reviewed in this volume (Dubielecka and Hoh). As AMD is a complex disease, genetic variation accounts for only a portion of risk; environmental influences also play an important role. The most consistently demonstrated factor conferring environmental risk is smoking (Chakravarthy, et al., 2007; de Jong, 2006). Drusen, focal circular deposits typically located in the macula, are the most commonly identified clinical feature of early AMD. They vary in size and distribution but are usually symmetric between the two eyes. These deposits, along with pigment alterations in the RPE (Figure 2A), increase the risk for development of more advanced stages of AMD associated with visual loss: geographic atrophy (GA) and choroidal neovascularization (CNV) (Figure 2B). Geographic atrophy is the default end-stage of vision loss due to AMD when CNV does not develop. It is recommended that patients with high-risk features (i.e. several medium to large drusen, RPE changes, and or CNV in the fellow eye) take age-related eye disease study

Geographic atrophy (GA) refers to a well-circumscribed area of RPE and photoreceptor cell loss in the macula associated with loss of retinal function that correlates clinically to dark spots (scotomas) in the vision. When involving the fovea, visual acuity declines depending on the size of the area affected. Visual loss from GA is indolent, but once present rates of GA progression occur up to 1.52 mm2/yr to 3.02 mm2/yr(Holz, et al., 2007; Lindblad, et al., 2009; Sunness, et al., 2007). There is currently no effective treatment for GA but several clinical

In contrast to GA, CNV is associated with more acute vision change that often presents to the ophthalmologist with metamorphopsia, scotoma, and/or decreased vision. Clinical signs include subretinal hemorrhage, lipid exudate, fluid, and a grey-green lesion (Figure 2B). Classically, dye leakage on fluorescein angiography confirms the growth of the new vessels that occur in the subRPE or subretinal space (Figure 2C). Optical coherence tomography (OCT) characterizes the presence of subretinal fluid, intraretinal edema, RPE detachment, or subRPE/retinal tissue from CNV (Figure 2D,E). Intravitreal injections of anti-VEGF agents are first-line therapy for most CNVs presenting to the retina specialist and almost always stabilize, if not improve, visual acuity.(Brown, et al., 2006; Martin, et al., 2011; Rosenfeld, et al., 2006; Tufail, et al., 2010) Because of its reliability and reproducibility, OCT is used to monitor the CNV and often guides the clinician in the decision-making process for

(AREDS)-formula vitamins to decrease the chance of CNV.(2001)

trials are underway.(Meleth, et al., 2011)

long-term treatment.(Fung, et al., 2007)

**degeneration** 

majority of the uveal blood supply (in some studies choroidal blood flow was estimated at more than 20x higher than in neural retina(Alm and Bill, 1973)). The proximity of the photoreceptor cells to the choroid is also required to remove wastes from the retina. Each RPE cell turns over many thousands of rod outer segment discs each day(Young, 1971) and this waste material from the RPE must be removed by the vasculature.

However, the juxtaposition of the choroid and retina poses a dilemma: while on one hand the photoreceptor cells have a major vascular requirement, on the other hand the microenvironment of the subretinal space is exquisitely regulated and the presence of subretinal fluid not inherent to this space (i.e., rhegmatogenous retinal detachment) results in rapid and severe vision loss. Having a dense vascular supply adjacent to a tissue that can not tolerate alterations in its interstitial space appears to be a recipe for disaster.

This dilemma is solved through the presence of soluble factors, such as PEDF(Dawson, et al., 1999; Ohno-Matsui, et al., 2001) and insoluble (structural) factors that keep the vasculature in check. In this chapter we will first briefly review the disease age-related macular degeneration (AMD) and then discuss the (normally) insoluble factors that guard the retina against vascular intrusion, namely Bruch's membrane (BrM), that play a role in preserving central vision.

Fig. 1. Section of Hadrian's wall near Carlisle, Cumbria, UK. While sections of stone have been plundered over the hundreds of years of its existence, the wall continues to have an imposing presence. Photo courtesy of Jenna M. Mullins.

majority of the uveal blood supply (in some studies choroidal blood flow was estimated at more than 20x higher than in neural retina(Alm and Bill, 1973)). The proximity of the photoreceptor cells to the choroid is also required to remove wastes from the retina. Each RPE cell turns over many thousands of rod outer segment discs each day(Young, 1971) and

However, the juxtaposition of the choroid and retina poses a dilemma: while on one hand the photoreceptor cells have a major vascular requirement, on the other hand the microenvironment of the subretinal space is exquisitely regulated and the presence of subretinal fluid not inherent to this space (i.e., rhegmatogenous retinal detachment) results in rapid and severe vision loss. Having a dense vascular supply adjacent to a tissue that can

This dilemma is solved through the presence of soluble factors, such as PEDF(Dawson, et al., 1999; Ohno-Matsui, et al., 2001) and insoluble (structural) factors that keep the vasculature in check. In this chapter we will first briefly review the disease age-related macular degeneration (AMD) and then discuss the (normally) insoluble factors that guard the retina against vascular intrusion, namely Bruch's membrane (BrM), that play a role in preserving central vision.

Fig. 1. Section of Hadrian's wall near Carlisle, Cumbria, UK. While sections of stone have been plundered over the hundreds of years of its existence, the wall continues to have an

imposing presence. Photo courtesy of Jenna M. Mullins.

this waste material from the RPE must be removed by the vasculature.

not tolerate alterations in its interstitial space appears to be a recipe for disaster.

#### **2. Clinical relevance of Bruch's membrane dysfunction: Age-related macular degeneration**

The most common ocular condition of elderly humans with BrM dysfunction is age-related macular degeneration (AMD). This disease affects the center of the vision by damaging the macula, the critical portion of the retina responsible for fine visual acuity and daily activities such as reading, driving and recognizing faces. It is the most common cause of blindness in the elderly population in the western world(Klein, et al., 1992; Mitchell, et al., 1995; Vingerling, et al., 1995). Genetic variants conferring risk for developing AMD include mutations in complement factor H (*CFH*), complement component 2 (*C2*), *C3, CFB*, and the age-related maculopathy susceptibility 2/HtrA serine peptidase 1 (*ARMS2/HTRA1*)(Bergeron-Sawitzke, et al., 2009; Edwards, et al., 2005; Fritsche, et al., 2008; Hageman, et al., 2005; Haines, et al., 2005; Kanda, et al., 2007; Klein, et al., 2005; Maller, et al., 2007; Rivera, et al., 2005; Yates, et al., 2007); also reviewed in this volume (Dubielecka and Hoh). As AMD is a complex disease, genetic variation accounts for only a portion of risk; environmental influences also play an important role. The most consistently demonstrated factor conferring environmental risk is smoking (Chakravarthy, et al., 2007; de Jong, 2006).

Drusen, focal circular deposits typically located in the macula, are the most commonly identified clinical feature of early AMD. They vary in size and distribution but are usually symmetric between the two eyes. These deposits, along with pigment alterations in the RPE (Figure 2A), increase the risk for development of more advanced stages of AMD associated with visual loss: geographic atrophy (GA) and choroidal neovascularization (CNV) (Figure 2B). Geographic atrophy is the default end-stage of vision loss due to AMD when CNV does not develop. It is recommended that patients with high-risk features (i.e. several medium to large drusen, RPE changes, and or CNV in the fellow eye) take age-related eye disease study (AREDS)-formula vitamins to decrease the chance of CNV.(2001)

Geographic atrophy (GA) refers to a well-circumscribed area of RPE and photoreceptor cell loss in the macula associated with loss of retinal function that correlates clinically to dark spots (scotomas) in the vision. When involving the fovea, visual acuity declines depending on the size of the area affected. Visual loss from GA is indolent, but once present rates of GA progression occur up to 1.52 mm2/yr to 3.02 mm2/yr(Holz, et al., 2007; Lindblad, et al., 2009; Sunness, et al., 2007). There is currently no effective treatment for GA but several clinical trials are underway.(Meleth, et al., 2011)

In contrast to GA, CNV is associated with more acute vision change that often presents to the ophthalmologist with metamorphopsia, scotoma, and/or decreased vision. Clinical signs include subretinal hemorrhage, lipid exudate, fluid, and a grey-green lesion (Figure 2B). Classically, dye leakage on fluorescein angiography confirms the growth of the new vessels that occur in the subRPE or subretinal space (Figure 2C). Optical coherence tomography (OCT) characterizes the presence of subretinal fluid, intraretinal edema, RPE detachment, or subRPE/retinal tissue from CNV (Figure 2D,E). Intravitreal injections of anti-VEGF agents are first-line therapy for most CNVs presenting to the retina specialist and almost always stabilize, if not improve, visual acuity.(Brown, et al., 2006; Martin, et al., 2011; Rosenfeld, et al., 2006; Tufail, et al., 2010) Because of its reliability and reproducibility, OCT is used to monitor the CNV and often guides the clinician in the decision-making process for long-term treatment.(Fung, et al., 2007)

Bruch's Membrane: The Critical Boundary in Macular Degeneration 53

The name "membrane" referring to a refractive band on histological sections has a long history in ophthalmology, and includes the membranes of Descemet and Bowman, as well as the descriptive internal and external limiting membranes. Since the invention of the transmission electron microscope, the term membrane became applied to much smaller

BrM itself is a multilayered, extracellular matrix compartment that includes two basement membranes (more appropriately called basal laminae) at its inner and outer aspects (Figure 3). In addition to the basal laminae of the RPE and choriocapillaris, BrM possesses a central layer of elastin surrounded by two layers of collagen. In normal physiology, BrM appears to

Fig. 3. Ultrastructure of murine (A) and human (B) Bruch's membrane. The basal laminae of the RPE (top) and choriocapillaris (bottom) are indicted by asterisks. Scale bar = 500nm (A) and 2µm (B). Labels indicate Bruch's membrane (bracket), the basal laminae (asterisks), the elastic lamina (EL), and RPE cell and the choriocapillaris lumen (CCL). Scale bar = 2µm. Left

Apart from its structural support function, BrM is also highly permeable to fluid and small molecules like oxygen and glucose. This quality likely derives from its sieve like structure appreciated by *en face* electron microscopy(Hogan, et al., 1971). Tracer studies in animals show that BrM (as well as the fenestrated choriocapillaris(Pino and Essner, 1981)) restrict the passage of even modestly sized proteins, while *in vitro* studies of excised human BrM can allow passage of molecules 200kDa or larger(Moore and Clover, 2001), recently discussed(Hussain, et al., 2010). Studies of hydraulic conductivity have invariably shown that the permeability of BrM decreases with increasing age (see below). RPE derived vascular endothelial growth factor also passes through BrM where it influences the structure

and function of the choriocapillaris(Saint-Geniez, et al., 2009).

structures (plasma membrane, nuclear membrane and basement membrane).

function as a robust barrier against neovascularization.

**3. Bruch's membrane: Anatomy** 

panel

The pathologic correlates of BrM changes in AMD is discussed further below. The next sections review the anatomy and early-onset consequences of changes to BrM.

Fig. 2. 87 year-old female presented with a one week history of metamorphopsia in the left eye and visual acuity of 20/80. (A) Fundus photo of the right eye demonstrating nonneovascular AMD. There are two medium sized drusen temporal to the fovea with few small drusen in the superotemporal arcade. RPE changes are most prominent just superior and nasal to the fovea. Visual acuity in this eye was 20/25. (B) The macula of the left eye has a deep gray-green lesion with subretinal hemorrhage consistent with neovascular AMD. (C) Fluorescein angiogram confirms leakage of the CNV centered in the temporal macula but involving the fovea. (D) Infrared image signifying the horizontal scan position of the spectral-domain OCT image of the left eye (E) through the fovea shows subfoveal material, intraretinal edema with a cyst along the temporal edge of the fovea and temporal subretinal fluid.

#### **3. Bruch's membrane: Anatomy**

52 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

The pathologic correlates of BrM changes in AMD is discussed further below. The next

Fig. 2. 87 year-old female presented with a one week history of metamorphopsia in the left eye and visual acuity of 20/80. (A) Fundus photo of the right eye demonstrating nonneovascular AMD. There are two medium sized drusen temporal to the fovea with few small drusen in the superotemporal arcade. RPE changes are most prominent just superior and nasal to the fovea. Visual acuity in this eye was 20/25. (B) The macula of the left eye has a deep gray-green lesion with subretinal hemorrhage consistent with neovascular AMD. (C) Fluorescein angiogram confirms leakage of the CNV centered in the temporal macula but involving the fovea. (D) Infrared image signifying the horizontal scan position of the spectral-domain OCT image of the left eye (E) through the fovea shows subfoveal material, intraretinal edema with a cyst along the temporal edge of the fovea and

temporal subretinal fluid.

sections review the anatomy and early-onset consequences of changes to BrM.

The name "membrane" referring to a refractive band on histological sections has a long history in ophthalmology, and includes the membranes of Descemet and Bowman, as well as the descriptive internal and external limiting membranes. Since the invention of the transmission electron microscope, the term membrane became applied to much smaller structures (plasma membrane, nuclear membrane and basement membrane).

BrM itself is a multilayered, extracellular matrix compartment that includes two basement membranes (more appropriately called basal laminae) at its inner and outer aspects (Figure 3). In addition to the basal laminae of the RPE and choriocapillaris, BrM possesses a central layer of elastin surrounded by two layers of collagen. In normal physiology, BrM appears to function as a robust barrier against neovascularization.

Fig. 3. Ultrastructure of murine (A) and human (B) Bruch's membrane. The basal laminae of the RPE (top) and choriocapillaris (bottom) are indicted by asterisks. Scale bar = 500nm (A) and 2µm (B). Labels indicate Bruch's membrane (bracket), the basal laminae (asterisks), the elastic lamina (EL), and RPE cell and the choriocapillaris lumen (CCL). Scale bar = 2µm. Left panel

Apart from its structural support function, BrM is also highly permeable to fluid and small molecules like oxygen and glucose. This quality likely derives from its sieve like structure appreciated by *en face* electron microscopy(Hogan, et al., 1971). Tracer studies in animals show that BrM (as well as the fenestrated choriocapillaris(Pino and Essner, 1981)) restrict the passage of even modestly sized proteins, while *in vitro* studies of excised human BrM can allow passage of molecules 200kDa or larger(Moore and Clover, 2001), recently discussed(Hussain, et al., 2010). Studies of hydraulic conductivity have invariably shown that the permeability of BrM decreases with increasing age (see below). RPE derived vascular endothelial growth factor also passes through BrM where it influences the structure and function of the choriocapillaris(Saint-Geniez, et al., 2009).

Bruch's Membrane: The Critical Boundary in Macular Degeneration 55

localized to some BrM deposits, as well as photoreceptor outer segments and retinal ganglion cells(Marmorstein, et al., 2002). Mice harboring one or more copies of the mutant *Efemp1* allele develop subRPE deposits resembling basal laminar deposit in human eyes(Fu, et al., 2007; Marmorstein, et al., 2007). In addition to the gene that encodes fibulin-3, mutations in the fibulin-5 gene, that also alter the secretion of the mutant protein(Lotery, et al., 2006), are associated with sporadic cases of AMD(Stone, et al., 2004). This protein regulates elastin assembly(Nakamura, et al., 2002; Yanagisawa, et al., 2002) and is localized to aging BrM and basal deposits in AMD eyes(Mullins, et al., 2007). Notably, fibulin-3 is a binding partner of tissue-inhibitor of metalloproteinase-3 (discussed more fully below)(Klenotic, et al., 2004), serving as a mechanistic link for neovascular membranes through TIMP-3 dysregulation in both conditions. Both fibulin-3 and fibulin-5 are widely expressed, and the reason for macular specific disease—and the pattern of drusen seen with EFEMP1 mutation—remains to be determined. Interestingly, the normal fibulin-5 protein inhibits angiogenesis, and its perturbation could remove a block to pathologic

*Mutations in TIMP3:* Another macular dystrophy, Sorsby fundus dystrophy, is caused by any of several mutations in the tissue inhibitor of metalloproteinases-3 gene, *TIMP3*(Weber, et al., 1994). Like fibulin-3, the TIMP3 protein is localized to BrM and is a major component of drusen(Fariss, et al., 1997). TIMP3 is capable of inhibiting VEGF-mediated angiogenesis by blocking the binding of VEGF to VEGF receptor-2 thereby inhibiting downstream signaling.(Qi, et al., 2003) Histopathology of eyes with Sorby fundus dystrophy shows dramatically thick, confluent deposits with a fragmented elastin layer of BrM (Capon et al., 1989; Chong et al., 2000). Bilateral subretinal neovascular membranes are frequently observed in patients with TIMP3 mutations by the fourth to fifth decades of life(Sivaprasad, et al., 2008; Sorsby and Mason, 1949). The most obvious explanation for grossly abnormal ECM deposits, as well as BrM breaks that permit neovasculrization, is that the TIMP-matrix metalloproteinase balance is disrupted, as TIMP3 substrates are metalloproteinases(Apte, et al., 1995). Interestingly, however, at least for one common TIMP3 mutation (Ser156Cys), the inhibition of MMP activity is not impaired compared to wild type TIMP3(Fogarasi, et al., 2008). Mouse models of SFD with dominant mutations have been generated and these animals show increased deposition of material at the level of BrM(Weber, et al., 2002).

Like the fibulins, TIMP3 is widely expressed(Apte, et al., 1994) and the reason for ocular specific disease is not clear, although it may relate to the unique relationship between the retina and choroidal vasculature. It is also notable that, in contrast to dominant mutations, mice that lack TIMP3 develop abnormal choroidal vasculature, and show loss of intercapillary pillars with large, sinusoidal vessels, indicating that TIMP3 normally functions in the development of the choroidal vasculature(Janssen, et al., 2008). Finally, it is notable that in a very large genome wide association study, an AMD susceptibility locus has been mapped to a region of chromosome 22 near the *TIMP3* gene(Chen, et al., 2011); the

*Mutations in ABCC6:* Whereas EFEMP1 and TIMP3 mutations have not as yet been associated with any pathology outside of the eye, mutations in *ABCC6* cause the systemic elastin disease, pseudoxanthoma elasticum (Bergen, et al., 2000; Le Saux, et al., 2000; Struk, et al., 2000).This disease affects elastic fibers in the skin and vasculature, in addition to BrM. The histopathologic phenotype shows breaks in BrM and elastin calcification and

functional impact of the variants at this locus are yet to be explored.

neovascularization(Albig and Schiemann, 2004).

Thus, in normal physiology, there is ready transport of water, nutrients, metabolites, retinoids, and other molecules necessary to maintain retinal health and function in both directions.

#### **4. Classes of Bruch's membrane molecules and their expression**

BrM is bounded by two basal laminae (belonging to the RPE and choriocapillaris respectively), which contain the normal array of basal lamina constituents (collagen type IV, laminin, heparan sulfate proteoglycan, and entactin/nidogen)(Kunze, et al., 2010). The specific collagen IV isoforms present within human(Chen, et al., 2003) and mouse(Bai, et al., 2009) eyes differ between the RPE and choriocapillary basal laminae.

Between the two basal laminae, two layers of fibrillar collagens, consisting of types I and III collagen, surround a central core of elastin.(Das, et al., 1990; Nakaizumi, 1964; Nakaizumi, et al., 1964b). The elastic layer, when viewed en face, shows a crisscrossing pattern of web-like fibers with spaces to permit diffusion and allow deformability. This layer has received attention due to its alterations in pathology and biological activity of its degradation products (see below). Elastic fibers in other tissues bind transforming growth factor beta(Karonen, et al., 1997) and endostatin(Miosge, et al., 1999), and a similar association of anti-angiogenic proteins with BrM elastin may therefore "repel" vascular growth into the subretinal space when the elastic layer is intact and when the stimulus for neovascularization is not overwhelming. It is easy to envision that loss of elastin, by whatever mechanism, could lead to removal of a structural and biochemical barrier against choroidal neovascularization. Elastin may also protect against complement mediated injury through its association with the complement regulator decay accelerating factor/CD55(Werth, et al., 1988).

In addition, numerous other ECM molecules have been noted in BrM including the antiangiogenic glycoprotein thrombospondin(Uno, et al., 2006) and sulfated proteoglycans(Call and Hollyfield, 1990; Clark, et al., 2011; Hewitt, et al., 1989). These molecules likely play important roles in hydration of the matrix, contribute to its barrier function(Prunte and Kain, 1995), the sequestration of growth factors, and may interact with the complement system (see below).

#### **5. Genetic variations affect Bruch's membrane and cause early-onset macular dystrophies**

The crucial role of ECM proteins in the macula is underscored by the fact that Bruch's membrane in this region is especially susceptible to mutations in genes with structural or regulatory roles in ECM metabolism. We briefly review a few of these familial macular diseases (i.e. macular dystrophies) with ECM abnormalities below.

*Mutations in fibulins:* Autosomal dominant radial drusen (also known as Malattia Leventinese, Doyne honeycomb retinal dystrophy, and dominant drusen) is a rare condition caused by a mutation (Arg345Trp) in the *EFEMP1* gene(Stone, et al., 1999). This gene encodes the ECM protein fibulin-3. Patients with this mutation typically have histologically unusual drusen like deposits that radiate from the macula, and may be complicated by neovascularization and/or atrophy(Michaelides, et al., 2006). The fibulin-3 protein is

Thus, in normal physiology, there is ready transport of water, nutrients, metabolites, retinoids, and other molecules necessary to maintain retinal health and function in both

BrM is bounded by two basal laminae (belonging to the RPE and choriocapillaris respectively), which contain the normal array of basal lamina constituents (collagen type IV, laminin, heparan sulfate proteoglycan, and entactin/nidogen)(Kunze, et al., 2010). The specific collagen IV isoforms present within human(Chen, et al., 2003) and mouse(Bai, et al.,

Between the two basal laminae, two layers of fibrillar collagens, consisting of types I and III collagen, surround a central core of elastin.(Das, et al., 1990; Nakaizumi, 1964; Nakaizumi, et al., 1964b). The elastic layer, when viewed en face, shows a crisscrossing pattern of web-like fibers with spaces to permit diffusion and allow deformability. This layer has received attention due to its alterations in pathology and biological activity of its degradation products (see below). Elastic fibers in other tissues bind transforming growth factor beta(Karonen, et al., 1997) and endostatin(Miosge, et al., 1999), and a similar association of anti-angiogenic proteins with BrM elastin may therefore "repel" vascular growth into the subretinal space when the elastic layer is intact and when the stimulus for neovascularization is not overwhelming. It is easy to envision that loss of elastin, by whatever mechanism, could lead to removal of a structural and biochemical barrier against choroidal neovascularization. Elastin may also protect against complement mediated injury through its association with the complement regulator decay accelerating

In addition, numerous other ECM molecules have been noted in BrM including the antiangiogenic glycoprotein thrombospondin(Uno, et al., 2006) and sulfated proteoglycans(Call and Hollyfield, 1990; Clark, et al., 2011; Hewitt, et al., 1989). These molecules likely play important roles in hydration of the matrix, contribute to its barrier function(Prunte and Kain, 1995), the sequestration of growth factors, and may interact with the complement

The crucial role of ECM proteins in the macula is underscored by the fact that Bruch's membrane in this region is especially susceptible to mutations in genes with structural or regulatory roles in ECM metabolism. We briefly review a few of these familial macular

*Mutations in fibulins:* Autosomal dominant radial drusen (also known as Malattia Leventinese, Doyne honeycomb retinal dystrophy, and dominant drusen) is a rare condition caused by a mutation (Arg345Trp) in the *EFEMP1* gene(Stone, et al., 1999). This gene encodes the ECM protein fibulin-3. Patients with this mutation typically have histologically unusual drusen like deposits that radiate from the macula, and may be complicated by neovascularization and/or atrophy(Michaelides, et al., 2006). The fibulin-3 protein is

**5. Genetic variations affect Bruch's membrane and cause early-onset** 

diseases (i.e. macular dystrophies) with ECM abnormalities below.

**4. Classes of Bruch's membrane molecules and their expression** 

2009) eyes differ between the RPE and choriocapillary basal laminae.

directions.

factor/CD55(Werth, et al., 1988).

system (see below).

**macular dystrophies** 

localized to some BrM deposits, as well as photoreceptor outer segments and retinal ganglion cells(Marmorstein, et al., 2002). Mice harboring one or more copies of the mutant *Efemp1* allele develop subRPE deposits resembling basal laminar deposit in human eyes(Fu, et al., 2007; Marmorstein, et al., 2007). In addition to the gene that encodes fibulin-3, mutations in the fibulin-5 gene, that also alter the secretion of the mutant protein(Lotery, et al., 2006), are associated with sporadic cases of AMD(Stone, et al., 2004). This protein regulates elastin assembly(Nakamura, et al., 2002; Yanagisawa, et al., 2002) and is localized to aging BrM and basal deposits in AMD eyes(Mullins, et al., 2007). Notably, fibulin-3 is a binding partner of tissue-inhibitor of metalloproteinase-3 (discussed more fully below)(Klenotic, et al., 2004), serving as a mechanistic link for neovascular membranes through TIMP-3 dysregulation in both conditions. Both fibulin-3 and fibulin-5 are widely expressed, and the reason for macular specific disease—and the pattern of drusen seen with EFEMP1 mutation—remains to be determined. Interestingly, the normal fibulin-5 protein inhibits angiogenesis, and its perturbation could remove a block to pathologic neovascularization(Albig and Schiemann, 2004).

*Mutations in TIMP3:* Another macular dystrophy, Sorsby fundus dystrophy, is caused by any of several mutations in the tissue inhibitor of metalloproteinases-3 gene, *TIMP3*(Weber, et al., 1994). Like fibulin-3, the TIMP3 protein is localized to BrM and is a major component of drusen(Fariss, et al., 1997). TIMP3 is capable of inhibiting VEGF-mediated angiogenesis by blocking the binding of VEGF to VEGF receptor-2 thereby inhibiting downstream signaling.(Qi, et al., 2003) Histopathology of eyes with Sorby fundus dystrophy shows dramatically thick, confluent deposits with a fragmented elastin layer of BrM (Capon et al., 1989; Chong et al., 2000). Bilateral subretinal neovascular membranes are frequently observed in patients with TIMP3 mutations by the fourth to fifth decades of life(Sivaprasad, et al., 2008; Sorsby and Mason, 1949). The most obvious explanation for grossly abnormal ECM deposits, as well as BrM breaks that permit neovasculrization, is that the TIMP-matrix metalloproteinase balance is disrupted, as TIMP3 substrates are metalloproteinases(Apte, et al., 1995). Interestingly, however, at least for one common TIMP3 mutation (Ser156Cys), the inhibition of MMP activity is not impaired compared to wild type TIMP3(Fogarasi, et al., 2008). Mouse models of SFD with dominant mutations have been generated and these animals show increased deposition of material at the level of BrM(Weber, et al., 2002).

Like the fibulins, TIMP3 is widely expressed(Apte, et al., 1994) and the reason for ocular specific disease is not clear, although it may relate to the unique relationship between the retina and choroidal vasculature. It is also notable that, in contrast to dominant mutations, mice that lack TIMP3 develop abnormal choroidal vasculature, and show loss of intercapillary pillars with large, sinusoidal vessels, indicating that TIMP3 normally functions in the development of the choroidal vasculature(Janssen, et al., 2008). Finally, it is notable that in a very large genome wide association study, an AMD susceptibility locus has been mapped to a region of chromosome 22 near the *TIMP3* gene(Chen, et al., 2011); the functional impact of the variants at this locus are yet to be explored.

*Mutations in ABCC6:* Whereas EFEMP1 and TIMP3 mutations have not as yet been associated with any pathology outside of the eye, mutations in *ABCC6* cause the systemic elastin disease, pseudoxanthoma elasticum (Bergen, et al., 2000; Le Saux, et al., 2000; Struk, et al., 2000).This disease affects elastic fibers in the skin and vasculature, in addition to BrM. The histopathologic phenotype shows breaks in BrM and elastin calcification and

Bruch's Membrane: The Critical Boundary in Macular Degeneration 57

Photoreceptor cells are extremely enriched for some classes of lipids, including the omega-3 fatty acid docosahexaenoic acid (DHA). This molecule can be metabolized to the neuroprotective molecule NPD1(Bazan, 2005) or oxidized to form a reactive, proinflammatory, angiogenic mediator carboxyethylpyrrole (CEP)(Hollyfield, et al., 2008). At least in the case of CEP, which is deposited in BrM in AMD(Gu, et al., 2003), photoreceptor outer segments are the most likely source. The photoreceptor origin of BrM lipids has been advanced for other classes as well(Pauleikhoff, et al., 1994). Some classes of lipids, including esterified and unesterified cholesterol(Haimovici, et al., 2001; Rudolf and Curcio, 2009), are assembled into distinct lipoprotein complexes and secreted by the RPE(Li, et al., 2005; Malek, et al., 2003; Wang, et al., 2011; Wang, et al., 2009). The provenance of all classes of lipids that accumulate in aging BrM is not entirely resolved, however, as recently

*Membrane attack complex of complement:* In addition to lipidic changes, activation of the complement system has received considerable attention, particularly in view of the compelling genetic evidence that polymorphisms in the complement inhibitor complement factor H (*CFH*) (in addition to other genes whose products regulate the complement system) (Bergeron-Sawitzke, et al., 2009; Edwards, et al., 2005; Fritsche, et al., 2008; Hageman, et al., 2005; Haines, et al., 2005; Kanda, et al., 2007; Klein, et al., 2005; Maller, et al., 2007; Rivera, et al., 2005; Yates, et al., 2007) is strongly associated with AMD (discussed above). Studies on the membrane attack complex of complement show that the majority of labeling is found in BrM and particularly around the choriocapillaris(Gerl, et al., 2002; Hageman, et al., 2005; Mullins, et al., 2011b; Seth, et al., 2008; Skeie, et al., 2010). In one study, this labeling was found to be statistically higher in aged (>69) as compared to young (<56) donors, indicating an increased complement load during aging(Seth, et al., 2008). The aberrant activation of the complement system may result in endothelial cell loss in the choriocapillaris(Mullins, et al., 2011b), increased VEGF synthesis by the RPE(Nozaki, et al., 2006), upregulated ICAM-1 RNA and protein by the choriocapillaris(Skeie, et al., 2010), and abnormal T cell activation(Liu, et al., 2011). The pathology that follows these challenges by the RPE and choroid are likely to worsen with aging. While there is generally not strong histological evidence that the membrane attack complex complement places a significant challenge to the RPE, it is noteworthy that expression of CD46 decreases in the pathogenesis of geographic atrophy(Vogt, et al., 2011). Since CD46 is a negative regulator of complement activity, playing a similar function at the cell surface that CFH plays in the extracellular space, even small or histochemically undetectable levels of complement could injure the

In addition to age, genotype of *CFH* appears to regulate the amount of membrane attack complex in the BrM/choriocapillaris. In a small study of genotyped eyes, we found that donors homozygous for the high risk allele (H at codon 402) had on average over 50% more membrane attack complex than age matched donors who were homozygous for the low risk allele(Mullins, et al., 2011a). Thus both genotype and age contribute to the deposition of

*Extracellular matrix:* ECM structural molecules also show age- and AMD-related changes. We will briefly review these changes, with special emphasis on elastin. Studies in mice show increased deposition of basal lamina proteins in BrM with age(Kunze, et al., 2010). While drusen themselves contain few classic extracellular matrix molecules(Hageman, et al., 1999;

potentially cytolytic membrane attack complexes in the aging choroid.

reviewed(Ebrahimi and Handa, 2011).

RPE in this weakened state.

fragmentation(Hagedoorn, 1939). The process of elastin calcification probably makes the brittle BrM susceptible to the fissures referred to clinically as angioid streaks, through which choroidal blood vessels invade the retina.

Thus, genetic conditions that affect the biochemistry of BrM can lead to a phenotype similar to end stage AMD.

#### **6. Bruch's membrane aging changes**

BrM undergoes age-related alterations that include anatomical, functional and molecular changes. We briefly review these below.

*Ultrastructural changes*: Anatomical changes in aging human BrM have been documented extensively and will not be covered to any significant degree here (see for example.(Feeney-Burns and Ellersieck, 1985; Hogan and Alvarado, 1967; Hogan, et al., 1971) These changes include the accumulation of membranous debris on both sides of the elastic lamina(Feeney-Burns and Ellersieck, 1985), accumulation of focal drusen between the RPE basal lamina and inner collagenous layer of BrM(Sarks, et al., 1999), and accumulation of more confluent deposits. These deposits may occupy the same space as drusen and exhibit the appearance of membranous debris (termed basal linear deposits) or may develop between the RPE cell and its basal lamina and have an amorphous or banded structure with a periodicity consistent with that of type VI collagen (referred to as basal laminar deposits)(Curcio and Millican, 1999; Knupp, et al., 2002; Nakaizumi, et al., 1964a; Sarks, et al., 2007). Changes in the elastic lamina are in their own section below.

*Functional changes:* In addition to the structural alterations that occur in BrM during aging, the function of BrM as a conduit for the passage of material between the RPE and choriocapillaris becomes substantially reduced. Functional changes in aging have been measured primarily through the use of Ussing chambers in which flow of tracer molecules can be assessed. A significant decrease in hydraulic conductivity—a measure of the permeability of tissue—has been described in the aging BrM(Hussain, et al., 2002; Moore, et al., 1995; Starita, et al., 1996). Hydraulic conductivity drops exponentially with advancing age, with greater than 50% reduction occurring every 20 years of age(Starita, et al., 1996). Sequential ablation of different layers of BrM suggests that the major site of resistance that differs between young and old eyes is the inner collagenous layer(Starita, et al., 1997) which also accumulates substantial debris in the aging macula(Newsome, et al., 1987b).

One probable function of elastin in BrM and the choroid is to structurally support this crucial tissue through the approximately 100,000 cycles of the choroidal pulse each day. Notably, in addition to loss of transport facility across BrM with age, a decline in the elasticity (approximately 1% per year after age 21) has also been reported(Ugarte, et al., 2006).

#### **7. Molecular changes in Bruch's membrane also occur during aging**

*Lipids*: An increased lipid content has been demonstrated using a variety of histochemical(Pauleikhoff, et al., 1990) and biochemical(Sheraidah, et al., 1993) approaches. Lipid content of BrM varies regionally between macular and extramacular samples(Gulcan, et al., 1993; Holz, et al., 1994).

fragmentation(Hagedoorn, 1939). The process of elastin calcification probably makes the brittle BrM susceptible to the fissures referred to clinically as angioid streaks, through which

Thus, genetic conditions that affect the biochemistry of BrM can lead to a phenotype similar

BrM undergoes age-related alterations that include anatomical, functional and molecular

*Ultrastructural changes*: Anatomical changes in aging human BrM have been documented extensively and will not be covered to any significant degree here (see for example.(Feeney-Burns and Ellersieck, 1985; Hogan and Alvarado, 1967; Hogan, et al., 1971) These changes include the accumulation of membranous debris on both sides of the elastic lamina(Feeney-Burns and Ellersieck, 1985), accumulation of focal drusen between the RPE basal lamina and inner collagenous layer of BrM(Sarks, et al., 1999), and accumulation of more confluent deposits. These deposits may occupy the same space as drusen and exhibit the appearance of membranous debris (termed basal linear deposits) or may develop between the RPE cell and its basal lamina and have an amorphous or banded structure with a periodicity consistent with that of type VI collagen (referred to as basal laminar deposits)(Curcio and Millican, 1999; Knupp, et al., 2002; Nakaizumi, et al., 1964a; Sarks, et al., 2007). Changes in

*Functional changes:* In addition to the structural alterations that occur in BrM during aging, the function of BrM as a conduit for the passage of material between the RPE and choriocapillaris becomes substantially reduced. Functional changes in aging have been measured primarily through the use of Ussing chambers in which flow of tracer molecules can be assessed. A significant decrease in hydraulic conductivity—a measure of the permeability of tissue—has been described in the aging BrM(Hussain, et al., 2002; Moore, et al., 1995; Starita, et al., 1996). Hydraulic conductivity drops exponentially with advancing age, with greater than 50% reduction occurring every 20 years of age(Starita, et al., 1996). Sequential ablation of different layers of BrM suggests that the major site of resistance that differs between young and old eyes is the inner collagenous layer(Starita, et al., 1997) which

One probable function of elastin in BrM and the choroid is to structurally support this crucial tissue through the approximately 100,000 cycles of the choroidal pulse each day. Notably, in addition to loss of transport facility across BrM with age, a decline in the elasticity (approximately 1% per year after age 21) has also been reported(Ugarte, et al.,

*Lipids*: An increased lipid content has been demonstrated using a variety of histochemical(Pauleikhoff, et al., 1990) and biochemical(Sheraidah, et al., 1993) approaches. Lipid content of BrM varies regionally between macular and extramacular samples(Gulcan,

also accumulates substantial debris in the aging macula(Newsome, et al., 1987b).

**7. Molecular changes in Bruch's membrane also occur during aging** 

choroidal blood vessels invade the retina.

**6. Bruch's membrane aging changes** 

the elastic lamina are in their own section below.

changes. We briefly review these below.

to end stage AMD.

2006).

et al., 1993; Holz, et al., 1994).

Photoreceptor cells are extremely enriched for some classes of lipids, including the omega-3 fatty acid docosahexaenoic acid (DHA). This molecule can be metabolized to the neuroprotective molecule NPD1(Bazan, 2005) or oxidized to form a reactive, proinflammatory, angiogenic mediator carboxyethylpyrrole (CEP)(Hollyfield, et al., 2008). At least in the case of CEP, which is deposited in BrM in AMD(Gu, et al., 2003), photoreceptor outer segments are the most likely source. The photoreceptor origin of BrM lipids has been advanced for other classes as well(Pauleikhoff, et al., 1994). Some classes of lipids, including esterified and unesterified cholesterol(Haimovici, et al., 2001; Rudolf and Curcio, 2009), are assembled into distinct lipoprotein complexes and secreted by the RPE(Li, et al., 2005; Malek, et al., 2003; Wang, et al., 2011; Wang, et al., 2009). The provenance of all classes of lipids that accumulate in aging BrM is not entirely resolved, however, as recently reviewed(Ebrahimi and Handa, 2011).

*Membrane attack complex of complement:* In addition to lipidic changes, activation of the complement system has received considerable attention, particularly in view of the compelling genetic evidence that polymorphisms in the complement inhibitor complement factor H (*CFH*) (in addition to other genes whose products regulate the complement system) (Bergeron-Sawitzke, et al., 2009; Edwards, et al., 2005; Fritsche, et al., 2008; Hageman, et al., 2005; Haines, et al., 2005; Kanda, et al., 2007; Klein, et al., 2005; Maller, et al., 2007; Rivera, et al., 2005; Yates, et al., 2007) is strongly associated with AMD (discussed above). Studies on the membrane attack complex of complement show that the majority of labeling is found in BrM and particularly around the choriocapillaris(Gerl, et al., 2002; Hageman, et al., 2005; Mullins, et al., 2011b; Seth, et al., 2008; Skeie, et al., 2010). In one study, this labeling was found to be statistically higher in aged (>69) as compared to young (<56) donors, indicating an increased complement load during aging(Seth, et al., 2008). The aberrant activation of the complement system may result in endothelial cell loss in the choriocapillaris(Mullins, et al., 2011b), increased VEGF synthesis by the RPE(Nozaki, et al., 2006), upregulated ICAM-1 RNA and protein by the choriocapillaris(Skeie, et al., 2010), and abnormal T cell activation(Liu, et al., 2011). The pathology that follows these challenges by the RPE and choroid are likely to worsen with aging. While there is generally not strong histological evidence that the membrane attack complex complement places a significant challenge to the RPE, it is noteworthy that expression of CD46 decreases in the pathogenesis of geographic atrophy(Vogt, et al., 2011). Since CD46 is a negative regulator of complement activity, playing a similar function at the cell surface that CFH plays in the extracellular space, even small or histochemically undetectable levels of complement could injure the RPE in this weakened state.

In addition to age, genotype of *CFH* appears to regulate the amount of membrane attack complex in the BrM/choriocapillaris. In a small study of genotyped eyes, we found that donors homozygous for the high risk allele (H at codon 402) had on average over 50% more membrane attack complex than age matched donors who were homozygous for the low risk allele(Mullins, et al., 2011a). Thus both genotype and age contribute to the deposition of potentially cytolytic membrane attack complexes in the aging choroid.

*Extracellular matrix:* ECM structural molecules also show age- and AMD-related changes. We will briefly review these changes, with special emphasis on elastin. Studies in mice show increased deposition of basal lamina proteins in BrM with age(Kunze, et al., 2010). While drusen themselves contain few classic extracellular matrix molecules(Hageman, et al., 1999;

Bruch's Membrane: The Critical Boundary in Macular Degeneration 59

et al., 2008). In addition, significantly elevated serum levels of elastin derived peptides (EDPs) have been found in the serum of CNV patients, with higher levels in CNV than in dry AMD, and higher levels in dry AMD than controls(Sivaprasad, et al., 2005). In this study, the authors noted that a sustained elevated level of EDPs could not be solely due to BrM degradation, and is instead likely to be due to systemic elastin abnormalities, consistent with those described by Blumenkranz et al. Finally, the breakdown products of elastin itself (elastin derived peptides) induce some angiogenic behaviors of choroidal endothelial cells(Skeie and Mullins, 2008)(Figure 4). Thus, the breakdown of elastin in BrM may simultaneously (a) remove a critical structural and chemical barrier to neovascularization and (b) actively induce the growth of pathologic blood vessels into the retina. Taken together, these studies provide strong support for the notion that abnormalities in elastin metabolism, both in ocular and extraocular tissues, are associated with the pathogenesis of

Fig. 4. Elastin monomers are crosslinked by lysyl oxidases into insoluble elastic networks that can repeatedly stretch and relax, essential for tissues like the choroid. Degradation of elastin by matrix metalloproteinases/elastases leads to the loss of integrity of the elastin network and the release of biologically active elastin derived peptides (EDPs) which can

The development of either excessive material in Bruch's membrane or erosion of Bruch's membrane may have serious consequences whether congenital or age-related. Some events, such as membrane attack complex deposition, may lead to choroidal endothelial cell loss(Mullins, et al., 2011b) and place ischemic stress on the oxygen hungry photoreceptor cells. Vascular loss may exacerbate the development of drusen, as the ability of the choroid to remove debris is further compromised; in this context it is notable that drusen form preferentially in eyes and in regions of eyes depleted of capillary lumens(Lengyel, et al.,

promote neovascularization. Adapted from Alberts et al., 2008

**8. Consequences of ECM abnormalities** 

2004; Mullins, et al., 2011b; Sarks, et al., 1999)(Figure 5).

neovascular AMD.

Newsome, et al., 1987a) aging BrM may show increased type IV collagen that contributes to its thickening(Marshall, et al., 1994). Basal lamina constituents are also present in granular basal laminar deposit material(van der Schaft, et al., 1994). In eyes with early AMD, the abundance of thrombospondin has been shown to be decreased(Uno, et al., 2006)—in view of the role of this protein in suppressing angiogenesis, its reduction may create a permissive environment for neovascularization. The altered homeostasis of ECM observed in aging and AMD may be in part due to the age-dependent reduction in matrix metalloproteinase activity(Guo, et al., 1999) and in the available pool of MMPs(Kumar, et al., 2010) that has been observed in human eyes. Proteomic studies of combined BrM/choroid layers from human donor eyes further reveal increases in some matrix proteins (e.g., MMP3, collagen I) and decreases in other proteins (e.g., nidogen-2, fibulin-1) during the progression of AMD(Yuan, et al., 2010).

Other age-related changes include the accumulation of iron and advanced glycation endproducts in BrM. While outside the scope of this article, the reader is referred to several excellent articles and reviews(Glenn, et al., 2009; Handa, et al., 1999; He, et al., 2007; Wong, et al., 2007), including in the current volume (Kaji et al.).

*Elastin metabolism in AMD:* Elastin is a hydrophobic glycoprotein of approximately 72kDa. It has an unusual primary sequence in which about one third of its amino acid composition is comprised of glycine. Unlike fibrillar collagens, which also contain glycines at every third amino acid, elastin does not form into triple helices. Instead, elastin monomers (tropoelastin molecules) are assembled into cross-linked networks by enzymatic modification of lysine residues by lysyl oxidases (Figure 4). The resulting network of hydrophobic elastin polymers is responsible for the elastic recoil of arteries, and indeed participates in maintenance of diastolic blood pressure(Faury, 2001). Elastin is found in multiple tissues including skin, large blood vessels, and BrM of the eye.

Several studies suggest that abnormal elastin physiology is involved in the pathogenesis of AMD, and especially neovascular AMD. One of the first indications of widespread elastin changes came from studies linking dermatologic changes with neovascular AMD. Whereas elastotic degeneration of sun-exposed skin is a typical finding, Blumenkranz and colleagues reported histopathologic evidence of elastotic degeneration of sun-protected skin in patients with CNV(Blumenkranz, et al., 1986). Second, Spraul and Grossniklaus, in performing morphometric studies on a series of human donor eyes with neovascular AMD, noted that calcification and fragmentation of the elastic lamina was a frequent finding(Spraul and Grossniklaus, 1997; Spraul, et al., 1999) suggesting elastin breakdown within BrM as a cause or consequence of AMD pathology. Third, Chong et al. made similar findings at the ultrastructural scale, in which we observed a relative paucity of macular elastin and noted that eyes with neovascular AMD tended to have a thinner and more porous elastic lamina of BrM than controls(Chong, et al., 2005) The thinning of the elastic lamina is in contrast to the general "thickening" of BrM during aging (which is actually accumulation of debris rather than expansion of its matrix components)(Feeney-Burns and Ellersieck, 1985). Fourth, genetic variations in genes associated with elastin biology are associated with macular disease, including *ABCC6* and *FBLN5* (discussed above) and polymorphisms in the elastin gene (*ELN*) that have been described in association with polypoidal choroidal vasculopathy(Kondo, et al., 2008). Mice deficient for the elastin crosslinking protein LOXL1 show disrupted BrM elastin and increased severity of experimental neovascularization(Yu,

Newsome, et al., 1987a) aging BrM may show increased type IV collagen that contributes to its thickening(Marshall, et al., 1994). Basal lamina constituents are also present in granular basal laminar deposit material(van der Schaft, et al., 1994). In eyes with early AMD, the abundance of thrombospondin has been shown to be decreased(Uno, et al., 2006)—in view of the role of this protein in suppressing angiogenesis, its reduction may create a permissive environment for neovascularization. The altered homeostasis of ECM observed in aging and AMD may be in part due to the age-dependent reduction in matrix metalloproteinase activity(Guo, et al., 1999) and in the available pool of MMPs(Kumar, et al., 2010) that has been observed in human eyes. Proteomic studies of combined BrM/choroid layers from human donor eyes further reveal increases in some matrix proteins (e.g., MMP3, collagen I) and decreases in other proteins (e.g., nidogen-2, fibulin-1) during the progression of

Other age-related changes include the accumulation of iron and advanced glycation endproducts in BrM. While outside the scope of this article, the reader is referred to several excellent articles and reviews(Glenn, et al., 2009; Handa, et al., 1999; He, et al., 2007; Wong,

*Elastin metabolism in AMD:* Elastin is a hydrophobic glycoprotein of approximately 72kDa. It has an unusual primary sequence in which about one third of its amino acid composition is comprised of glycine. Unlike fibrillar collagens, which also contain glycines at every third amino acid, elastin does not form into triple helices. Instead, elastin monomers (tropoelastin molecules) are assembled into cross-linked networks by enzymatic modification of lysine residues by lysyl oxidases (Figure 4). The resulting network of hydrophobic elastin polymers is responsible for the elastic recoil of arteries, and indeed participates in maintenance of diastolic blood pressure(Faury, 2001). Elastin is found in multiple tissues

Several studies suggest that abnormal elastin physiology is involved in the pathogenesis of AMD, and especially neovascular AMD. One of the first indications of widespread elastin changes came from studies linking dermatologic changes with neovascular AMD. Whereas elastotic degeneration of sun-exposed skin is a typical finding, Blumenkranz and colleagues reported histopathologic evidence of elastotic degeneration of sun-protected skin in patients with CNV(Blumenkranz, et al., 1986). Second, Spraul and Grossniklaus, in performing morphometric studies on a series of human donor eyes with neovascular AMD, noted that calcification and fragmentation of the elastic lamina was a frequent finding(Spraul and Grossniklaus, 1997; Spraul, et al., 1999) suggesting elastin breakdown within BrM as a cause or consequence of AMD pathology. Third, Chong et al. made similar findings at the ultrastructural scale, in which we observed a relative paucity of macular elastin and noted that eyes with neovascular AMD tended to have a thinner and more porous elastic lamina of BrM than controls(Chong, et al., 2005) The thinning of the elastic lamina is in contrast to the general "thickening" of BrM during aging (which is actually accumulation of debris rather than expansion of its matrix components)(Feeney-Burns and Ellersieck, 1985). Fourth, genetic variations in genes associated with elastin biology are associated with macular disease, including *ABCC6* and *FBLN5* (discussed above) and polymorphisms in the elastin gene (*ELN*) that have been described in association with polypoidal choroidal vasculopathy(Kondo, et al., 2008). Mice deficient for the elastin crosslinking protein LOXL1 show disrupted BrM elastin and increased severity of experimental neovascularization(Yu,

AMD(Yuan, et al., 2010).

et al., 2007), including in the current volume (Kaji et al.).

including skin, large blood vessels, and BrM of the eye.

et al., 2008). In addition, significantly elevated serum levels of elastin derived peptides (EDPs) have been found in the serum of CNV patients, with higher levels in CNV than in dry AMD, and higher levels in dry AMD than controls(Sivaprasad, et al., 2005). In this study, the authors noted that a sustained elevated level of EDPs could not be solely due to BrM degradation, and is instead likely to be due to systemic elastin abnormalities, consistent with those described by Blumenkranz et al. Finally, the breakdown products of elastin itself (elastin derived peptides) induce some angiogenic behaviors of choroidal endothelial cells(Skeie and Mullins, 2008)(Figure 4). Thus, the breakdown of elastin in BrM may simultaneously (a) remove a critical structural and chemical barrier to neovascularization and (b) actively induce the growth of pathologic blood vessels into the retina. Taken together, these studies provide strong support for the notion that abnormalities in elastin metabolism, both in ocular and extraocular tissues, are associated with the pathogenesis of neovascular AMD.

Fig. 4. Elastin monomers are crosslinked by lysyl oxidases into insoluble elastic networks that can repeatedly stretch and relax, essential for tissues like the choroid. Degradation of elastin by matrix metalloproteinases/elastases leads to the loss of integrity of the elastin network and the release of biologically active elastin derived peptides (EDPs) which can promote neovascularization. Adapted from Alberts et al., 2008

#### **8. Consequences of ECM abnormalities**

The development of either excessive material in Bruch's membrane or erosion of Bruch's membrane may have serious consequences whether congenital or age-related. Some events, such as membrane attack complex deposition, may lead to choroidal endothelial cell loss(Mullins, et al., 2011b) and place ischemic stress on the oxygen hungry photoreceptor cells. Vascular loss may exacerbate the development of drusen, as the ability of the choroid to remove debris is further compromised; in this context it is notable that drusen form preferentially in eyes and in regions of eyes depleted of capillary lumens(Lengyel, et al., 2004; Mullins, et al., 2011b; Sarks, et al., 1999)(Figure 5).

Bruch's Membrane: The Critical Boundary in Macular Degeneration 61

the complement system and summarized in recent reviews. (Yehoshua et al., 2011; Meleth et al., 2011). Ameliorating AMD by modulating other changes that occur in the ECM remains to be explored. This discussion is not intended to be inclusive but to highlight a few areas in

As discussed above, aberrant elastin metabolism and perhaps signaling are associated with AMD, especially neovascular AMD. Systemic elastin abnormalities occur in AMD, and the local changes in BrM likely at once both remove a barrier to neovascualization and promote growth of new blood vessels from the choroid. A better understanding of the molecules involved in this signaling may provide additional targets of neovascular AMD to accompany anti-VEGF drugs in some cases. This is vital in the cases of neovascular AMD that do not fully respond to anti-VEGF therapy or that develop 'tachyphylaxis' to anti-VEGF

In addition to the pro-angiogenic effects of the elastin components of BrM, other components of BrM are often anti-angiogenic. This has been noted for several molecules including thrombospondin, as discussed above, endostatin(Marneros, et al., 2007; Mori, et al., 2001) a fragment of collagen type XVIII, and fragments of collagen IV(Lima, et al., 2006). Enhancing the expression of these anti-angiogenic molecules through gene delivery or systemic administration shows promise as another tool against neovascular AMD, especially since decreased expression of these inhibitors occurs during pathogenesis(Bhutto, et al., 2008). A caution for these studies is that increases in BrM collagen IV have been linked to thickening of BrM and the development of subRPE

Apart from interfering with signaling events in the aging macula, it may also be possible and necessary to reconstruct BrM in some cases. With the promise of stem cell mediated therapies for AMD and other maculopathies, having a substrate on which transplanted cells can attach and perform their normal physiologic functions is a considerable challenge. Replacing defective RPE cells has been proposed as a mechanism to ameliorate both neovascular(Tezel, et al., 2007) and atrophic(Du, et al., 2011) AMD. Elegant experiments at delivering RPE cells from a variety of potential sources(da Cruz, et al., 2007) indicate that modifying BrM to accept transplanted cells(Gullapalli, et al., 2004) and/or delivering cells on degradable scaffolds(Thomson, et al., 2010) will be necessary for successful transplantation. Advances in combining materials science with cell biology will be essential

In summary, the relationship between the photoreceptor cells/RPE and the vascular supply is complex, and the intervening layer of extracellular matrix is especially susceptible to genetic and age-related changes that impair its function. A better understanding of this

RPE, retinal pigment epithelium; EC, endothelial cell; BrM, Bruch's membrane; ECM, extracellular matrix; AMD, age-related macular degeneration; GA, geographic atrophy; CNV, choroidal neovascularization; OCT, optical coherence tomography; VEGF, vascular

which a better understanding of ECM pathobiology of AMD may be helpful.

drugs.(Gasperini, et al., 2011; Schaal, et al., 2008)

for the next generation of treatments for AMD.

complex boundary will provide novel opportunities for therapy.

deposits, as noted above.

**10. Abbreviations** 

endothelial growth factor

In addition, the interposition of lipid rich material between the retina and its vascular supply, even with a healthy choriocapillaris, can compromise the normal trafficking between these tissues(Curcio, et al., 2010). The accumulation of lipids in BrM (consistent with the reduced ability to move water through the aging BrM--discussed above) has led some investigators to the attractive hypothesis that normal pumping of fluid through the RPE, combined with a nonpermeable BrM, could cause pigment epithelial detachments(Pauleikhoff, et al., 1990). Moreover, the molecules that accumulate in aging Bruch's membrane—including CEP-modified proteins and advanced glycation endproducts—may themselves be toxic, pro-inflammatory, and pro-angiogenic(Ebrahem, et al., 2006; Glenn, et al., 2009; Ma, et al., 2007).

Fig. 5. In human eyes, formation of deposits in the ECM, such as drusen (asterisks) occur preferentially in areas of choroid without capillary lumens, suggesting that the clearance of extracellular debris by the choriocapillaris may be preventative against drusen formation. Arrows, indicate "ghost" capillary vessels. Green immunoreactivity, anti-elastin; red labeling, UEA-I (a vascular marker); orange autofluorescence in the RPE is due to lipofuscin; blue fluorescence is due to a nuclear counterstain. Scale bar = 50µm.

While atrophic changes in AMD may be most easily attributed to excess material deposited in BrM, at some point in the development of neovascular AMD the structure of BrM becomes compromised. That defects in BrM permit angiogenesis into the retina is clear from both genetic diseases in which large cracks appear in the calcified BrM (i.e., pseudoxanthoma elasticum, discussed above) as well as animal models of CNV in which rupture of BrM is sufficient to induce neovascular AMD-like pathology(Ryan, 1980). The molecular changes in early and late AMD that include loss of anti-angiogenic proteins(Bhutto, et al., 2008) and calcification and fragmentation of BrM elastin(Chong, et al., 2005; Spraul and Grossniklaus, 1997) show that breakdown of BrM elastin occurs during the progression of AMD and provides opportunities for neovascular events. These may be especially problematic since, as noted above, fragments of elastin are sufficient to promote the migration of choroidal EC(Skeie and Mullins, 2008).

#### **9. Potential ECM mediated therapeutics**

In view of the many roles of the extracellular matrix in macular health and disease, several potential areas exist for ameliorating the pathogenesis of AMD. Current early-phase clinical trials for non-neovascular AMD directed at extracellular matrix dysfunction are limited to

In addition, the interposition of lipid rich material between the retina and its vascular supply, even with a healthy choriocapillaris, can compromise the normal trafficking between these tissues(Curcio, et al., 2010). The accumulation of lipids in BrM (consistent with the reduced ability to move water through the aging BrM--discussed above) has led some investigators to the attractive hypothesis that normal pumping of fluid through the RPE, combined with a nonpermeable BrM, could cause pigment epithelial detachments(Pauleikhoff, et al., 1990). Moreover, the molecules that accumulate in aging Bruch's membrane—including CEP-modified proteins and advanced glycation endproducts—may themselves be toxic, pro-inflammatory, and pro-angiogenic(Ebrahem, et

Fig. 5. In human eyes, formation of deposits in the ECM, such as drusen (asterisks) occur preferentially in areas of choroid without capillary lumens, suggesting that the clearance of extracellular debris by the choriocapillaris may be preventative against drusen formation. Arrows, indicate "ghost" capillary vessels. Green immunoreactivity, anti-elastin; red

labeling, UEA-I (a vascular marker); orange autofluorescence in the RPE is due to lipofuscin;

While atrophic changes in AMD may be most easily attributed to excess material deposited in BrM, at some point in the development of neovascular AMD the structure of BrM becomes compromised. That defects in BrM permit angiogenesis into the retina is clear from both genetic diseases in which large cracks appear in the calcified BrM (i.e., pseudoxanthoma elasticum, discussed above) as well as animal models of CNV in which rupture of BrM is sufficient to induce neovascular AMD-like pathology(Ryan, 1980). The molecular changes in early and late AMD that include loss of anti-angiogenic proteins(Bhutto, et al., 2008) and calcification and fragmentation of BrM elastin(Chong, et al., 2005; Spraul and Grossniklaus, 1997) show that breakdown of BrM elastin occurs during the progression of AMD and provides opportunities for neovascular events. These may be especially problematic since, as noted above, fragments of elastin are sufficient to promote

In view of the many roles of the extracellular matrix in macular health and disease, several potential areas exist for ameliorating the pathogenesis of AMD. Current early-phase clinical trials for non-neovascular AMD directed at extracellular matrix dysfunction are limited to

blue fluorescence is due to a nuclear counterstain. Scale bar = 50µm.

the migration of choroidal EC(Skeie and Mullins, 2008).

**9. Potential ECM mediated therapeutics** 

al., 2006; Glenn, et al., 2009; Ma, et al., 2007).

the complement system and summarized in recent reviews. (Yehoshua et al., 2011; Meleth et al., 2011). Ameliorating AMD by modulating other changes that occur in the ECM remains to be explored. This discussion is not intended to be inclusive but to highlight a few areas in which a better understanding of ECM pathobiology of AMD may be helpful.

As discussed above, aberrant elastin metabolism and perhaps signaling are associated with AMD, especially neovascular AMD. Systemic elastin abnormalities occur in AMD, and the local changes in BrM likely at once both remove a barrier to neovascualization and promote growth of new blood vessels from the choroid. A better understanding of the molecules involved in this signaling may provide additional targets of neovascular AMD to accompany anti-VEGF drugs in some cases. This is vital in the cases of neovascular AMD that do not fully respond to anti-VEGF therapy or that develop 'tachyphylaxis' to anti-VEGF drugs.(Gasperini, et al., 2011; Schaal, et al., 2008)

In addition to the pro-angiogenic effects of the elastin components of BrM, other components of BrM are often anti-angiogenic. This has been noted for several molecules including thrombospondin, as discussed above, endostatin(Marneros, et al., 2007; Mori, et al., 2001) a fragment of collagen type XVIII, and fragments of collagen IV(Lima, et al., 2006). Enhancing the expression of these anti-angiogenic molecules through gene delivery or systemic administration shows promise as another tool against neovascular AMD, especially since decreased expression of these inhibitors occurs during pathogenesis(Bhutto, et al., 2008). A caution for these studies is that increases in BrM collagen IV have been linked to thickening of BrM and the development of subRPE deposits, as noted above.

Apart from interfering with signaling events in the aging macula, it may also be possible and necessary to reconstruct BrM in some cases. With the promise of stem cell mediated therapies for AMD and other maculopathies, having a substrate on which transplanted cells can attach and perform their normal physiologic functions is a considerable challenge. Replacing defective RPE cells has been proposed as a mechanism to ameliorate both neovascular(Tezel, et al., 2007) and atrophic(Du, et al., 2011) AMD. Elegant experiments at delivering RPE cells from a variety of potential sources(da Cruz, et al., 2007) indicate that modifying BrM to accept transplanted cells(Gullapalli, et al., 2004) and/or delivering cells on degradable scaffolds(Thomson, et al., 2010) will be necessary for successful transplantation. Advances in combining materials science with cell biology will be essential for the next generation of treatments for AMD.

In summary, the relationship between the photoreceptor cells/RPE and the vascular supply is complex, and the intervening layer of extracellular matrix is especially susceptible to genetic and age-related changes that impair its function. A better understanding of this complex boundary will provide novel opportunities for therapy.

#### **10. Abbreviations**

RPE, retinal pigment epithelium; EC, endothelial cell; BrM, Bruch's membrane; ECM, extracellular matrix; AMD, age-related macular degeneration; GA, geographic atrophy; CNV, choroidal neovascularization; OCT, optical coherence tomography; VEGF, vascular endothelial growth factor

Bruch's Membrane: The Critical Boundary in Macular Degeneration 63

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#### **11. Acknowledgments**

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**4** 

*Japan* 

**Non-Enzymatic Post-Translational** 

Yuichi Kaji1, Tetsuro Oshika1 and Noriko Fujii2

**Modifications in the Development of Age-Related Macular Degeneration** 

*1Department of Pathophysiology of Vision and Ophthalmology, University of Tsukuba* 

Age-related macular degeneration (AMD) is a leading cause of blindness among Caucasians in various countries.1-3 In addition, cases of AMD are increasing among non-Caucasians, and AMD has become one of the major causes of blindness worldwide. To address this problem, several etiological, pathological, and basic science studies are being conducted. Etiological studies have revealed that the risk factors of AMD include smoking,4 increasing age, and the presence of cardiovascular disorders.5 However, the molecular mechanisms

Pathoclinical studies have revealed macular drusen, which are small lumps of abnormally accumulated proteins beneath the retinal pigment of epithelial cells, to be a sign of AMD (Figure 1).6 Proteomics analysis of drusen has revealed that it is composed of various proteins, including clusterin, albumin, TIMP3, vitronectin, complement components, and crystallin.

However, the pathological role of drusen in the development of AMD is still unclear.

Fig. 1. **Drusen as an early sign of age-related macular degeneration** 

Drusen are seen as yellow to white materials especially in the macular area (Left).

pigmented epithelium (RPE) and Bruch's membrane (arrows in the right Fig.).

Histologically, drusen are recognized as extracellular deposits that form between the retinal

**1. Introduction** 

linking these risk factors to AMD are still unclear.

*Graduate School of Comprehensive and Human Scicences, Tsukuba, Ibaraki, 2Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka,* 


### **Non-Enzymatic Post-Translational Modifications in the Development of Age-Related Macular Degeneration**

Yuichi Kaji1, Tetsuro Oshika1 and Noriko Fujii2 *1Department of Pathophysiology of Vision and Ophthalmology, University of Tsukuba Graduate School of Comprehensive and Human Scicences, Tsukuba, Ibaraki, 2Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka, Japan* 

#### **1. Introduction**

72 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Yates JR, Sepp T, Matharu BK, Khan JC, Thurlby DA, Shahid H, Clayton DG, Hayward C,

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proteomics: comparison of the macular Bruch membrane/choroid complex from age-related macular degeneration and normal eyes. Mol Cell Proteomics 9(6):1031-

> Age-related macular degeneration (AMD) is a leading cause of blindness among Caucasians in various countries.1-3 In addition, cases of AMD are increasing among non-Caucasians, and AMD has become one of the major causes of blindness worldwide. To address this problem, several etiological, pathological, and basic science studies are being conducted. Etiological studies have revealed that the risk factors of AMD include smoking,4 increasing age, and the presence of cardiovascular disorders.5 However, the molecular mechanisms linking these risk factors to AMD are still unclear.

> Pathoclinical studies have revealed macular drusen, which are small lumps of abnormally accumulated proteins beneath the retinal pigment of epithelial cells, to be a sign of AMD (Figure 1).6 Proteomics analysis of drusen has revealed that it is composed of various proteins, including clusterin, albumin, TIMP3, vitronectin, complement components, and crystallin. However, the pathological role of drusen in the development of AMD is still unclear.

#### Fig. 1. **Drusen as an early sign of age-related macular degeneration**

Drusen are seen as yellow to white materials especially in the macular area (Left). Histologically, drusen are recognized as extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane (arrows in the right Fig.).

Non-Enzymatic Post-Translational

**2.1 Advanced glycation end products** 

Fig. 3. **Multistep reactions of AGE formation** 

retinopathy and keratopathy, respectively.11-14

**2.2 D-amino acids** 

Modifications in the Development of Age-Related Macular Degeneration 75

Advanced glycation end products (AGEs) are the final reaction products of proteins and reducing sugars. The reaction of reducing sugars (glucose and fructose) with Lys and Arg residues in proteins leads to the formation of Schiff base and Amadori products, which slowly undergo oxidation, dehydration, and condensation to form AGEs (Figure 3). The final products vary depending on the proteins, sugars, and the reactions involved. However, common structures called AGE motifs are seen in the products, irrespective of the proteins

Proteins and reducing sugars react with each other to form Amadori products. After multistep

Our body is like an incubator containing sugars and proteins, in which AGEs are naturally formed, particularly in the tissues with a low turnover rate such as bone, teeth, dura mater, and lens. AGEs tend to accumulate with age, and they are known to do so in the target organ of age-related disorders such as Alzheimer's disease and atherosclerosis. Thus,

Formation of AGEs increases in diabetes partly because the concentration of reducing sugars increases in the blood. An accumulation of AGEs is seen in the thickened basement membrane and sclerotic lesions of the glomerulus in diabetic nephropathy. Furthermore, AGEs in the vitreous body and cornea are involved in the development of diabetic

AGEs, along with being the accessory products of the aging process and diabetes, also possibly contribute to the aging process and diabetic complications. AGEs are known to alter the structure of proteins by intra- and inter-molecular crosslinking, consequently disrupting their function. For example, naïve laminin has a figure of the cross, but AGEmodified laminin is deformed, and thus, loses its adhesion property to epithelial cells.

Proteins of all living organism on earth are composed of 20 types of amino acids. Among them, 19 amino acids other than glycine have chiral carbon atoms in the molecule.

reactions, including oxidation, dehydration, and condensation, AGEs are generated.

accumulation of AGEs in tissues is thought to be a biomarker of the aging process.


and sugars involved. Recently, a number of AGE motifs, including *N*<sup>ε</sup>

lysine, imidazoline, pyrraline, and pentosidine, have been revealed.

AMD and other aging-related changes in the body have certain common characteristics, including the aggregation of abnormal proteins, seen in the lens and brain of patients with cataract and Alzheimer's disease, respectively. Post-translational modifications of proteins are also aging-related changes commonly seen in the target organ. For example, advanced glycation of proteins and racemization of amino acids with resultant D-amino acid formation in proteins are well-documented changes related to aging. Post-translational modification of proteins occurs because of aging, and it contributes to the aging changes of organs.

In order to elucidate the molecular mechanism of AMD, we evaluated the role of posttranslational modifications of proteins in the development of AMD, particularly the formation of advanced glycation end products (AGEs) and D-amino acids in the development of drusen.

### **2. Post-translational modifications in age-related disorders**

Post-translational modification of proteins is a molecular characteristic of the aging process. This is of 2 types as follows: enzymatic post-translational modification, which includes phosphorylation and glycosylation and is essential for protein function; and non-enzymatic post-translational modification that includes advanced glycation, racemization (and the resultant D-amino acid formation), and truncation, which impairs protein function, contributing to the aging process at the molecular level in various organs (Figure 2).

#### Fig. 2. **Aging of proteins at the molecular level**

Non-enzymatic post-translational modifications of proteins, including formation of AGEs and D-amino acids, are recognized as an aging process of proteins at the molecular level.

#### **2.1 Advanced glycation end products**

74 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

AMD and other aging-related changes in the body have certain common characteristics, including the aggregation of abnormal proteins, seen in the lens and brain of patients with cataract and Alzheimer's disease, respectively. Post-translational modifications of proteins are also aging-related changes commonly seen in the target organ. For example, advanced glycation of proteins and racemization of amino acids with resultant D-amino acid formation in proteins are well-documented changes related to aging. Post-translational modification of

In order to elucidate the molecular mechanism of AMD, we evaluated the role of posttranslational modifications of proteins in the development of AMD, particularly the formation of advanced glycation end products (AGEs) and D-amino acids in the

Post-translational modification of proteins is a molecular characteristic of the aging process. This is of 2 types as follows: enzymatic post-translational modification, which includes phosphorylation and glycosylation and is essential for protein function; and non-enzymatic post-translational modification that includes advanced glycation, racemization (and the resultant D-amino acid formation), and truncation, which impairs protein function,

contributing to the aging process at the molecular level in various organs (Figure 2).

Non-enzymatic post-translational modifications of proteins, including formation of AGEs and D-amino acids, are recognized as an aging process of proteins at the molecular level.

proteins occurs because of aging, and it contributes to the aging changes of organs.

**2. Post-translational modifications in age-related disorders** 

Fig. 2. **Aging of proteins at the molecular level** 

development of drusen.

Advanced glycation end products (AGEs) are the final reaction products of proteins and reducing sugars. The reaction of reducing sugars (glucose and fructose) with Lys and Arg residues in proteins leads to the formation of Schiff base and Amadori products, which slowly undergo oxidation, dehydration, and condensation to form AGEs (Figure 3). The final products vary depending on the proteins, sugars, and the reactions involved. However, common structures called AGE motifs are seen in the products, irrespective of the proteins and sugars involved. Recently, a number of AGE motifs, including *N*<sup>ε</sup> -(carboxy) methyl-Llysine, imidazoline, pyrraline, and pentosidine, have been revealed.

#### Fig. 3. **Multistep reactions of AGE formation**

Proteins and reducing sugars react with each other to form Amadori products. After multistep reactions, including oxidation, dehydration, and condensation, AGEs are generated.

Our body is like an incubator containing sugars and proteins, in which AGEs are naturally formed, particularly in the tissues with a low turnover rate such as bone, teeth, dura mater, and lens. AGEs tend to accumulate with age, and they are known to do so in the target organ of age-related disorders such as Alzheimer's disease and atherosclerosis. Thus, accumulation of AGEs in tissues is thought to be a biomarker of the aging process.

Formation of AGEs increases in diabetes partly because the concentration of reducing sugars increases in the blood. An accumulation of AGEs is seen in the thickened basement membrane and sclerotic lesions of the glomerulus in diabetic nephropathy. Furthermore, AGEs in the vitreous body and cornea are involved in the development of diabetic retinopathy and keratopathy, respectively.11-14

AGEs, along with being the accessory products of the aging process and diabetes, also possibly contribute to the aging process and diabetic complications. AGEs are known to alter the structure of proteins by intra- and inter-molecular crosslinking, consequently disrupting their function. For example, naïve laminin has a figure of the cross, but AGEmodified laminin is deformed, and thus, loses its adhesion property to epithelial cells.

#### **2.2 D-amino acids**

Proteins of all living organism on earth are composed of 20 types of amino acids. Among them, 19 amino acids other than glycine have chiral carbon atoms in the molecule.

Non-Enzymatic Post-Translational

years had drusen. Antibodies: *N*<sup>ε</sup>

prepared and purified.

solution dissolved in phosphate-buffered saline.

Japan).

Modifications in the Development of Age-Related Macular Degeneration 77

Eye samples: Nine eyes from 9 donors of 18 to 88 years of age were obtained at the time of necropsy and used as samples for this study. Among them, 4 eyes from donors older than 68

body. A monoclonal antibody to CML was purchased (Transgenic Co. Ltd, Kumamoto,

The preparation and characterization of the primary antibody of D-β-Asp containing proteins was as described previously.27 The polyclonal antibody to the synthetic peptide called peptide 3R(Gly-Leu-D-β-Asp-Ala-Thy-Gly-Leu-D-β-Asp-Ala-Thy-Gly-Leu-D-β-Asp-Ala-Thy) corresponding to 3 repeats of positions 149–153 of the human α-A-crystallin was

Immunohistochemistry: Immunohistochemical localization of CML and D-β-Asp containing proteins was investigated using the antibodies mentioned above. After fixation with 10% formalin solution, 4-μm-thick sections of the paraffin-embedded ocular samples were prepared. After deparaffinization, the sections were treated with 2 mg/mL of monoclonal antibody to CML or 1:500 diluted polyclonal antibody to D-β-Asp-containing proteins. After washing with phosphate-buffered saline, the sections were treated with secondary antibodies labeled with a polymer of horse radish peroxidase (Hitofine, Max-PO kit, Nichirei Co. Ltd, Tokyo, Japan). The final products were visualized using diaminobenzidine

No immunoreactivity to CML was seen in the retinas, choroids, or scleras of 5 eyes of donors younger than 18 years of age. In contrast, moderate immunoreactivity was seen in the retinal nerve fiber layers, and strong immunoreactivity was seen in the drusen and the

thickened Bruch's membrane of the 5 eyes of donors older than 68 years (Figure 5).

Fig. 5. **Immunohistochemical localization of AGE in human retina and choroid** 

(arrows), and thickened Bruch's membrane (arrowheads).

No immunoreactivity to CML, a major component of AGEs, is noted in young donor eyes. In contrast, immunoreactivity to CML is seen in the retinal nerve fiber layer (\*), drusen


Depending on the configuration of the side chains around the chiral carbon atoms, amino acids are either l- or d-amino acids. If amino acids were synthesized chemically, the quantity of l- and d-amino acids would be equal. However, proteins in all living organisms are composed exclusively of l-amino acids.

L-amino acids are converted to D-amino acids with a half-life of several thousand years, thus creating an equal amount of L- and D-amino acids over a particular period. This amount of D-amino acids is used for the age determination of fossils. Thus, D-amino acids are regarded as the products of post-mortal change and are irrelevant to living organisms. However, biologically uncommon D-amino acids that are enantiomers of L-amino acids have been found in the lenses16-19, teeth20, bones, brains21, skin22, aortas23, erythrocytes24, lungs25, and ligaments of elderly donors.26 The presence of D-amino acids in aged tissues of the living body is considered to be a result of racemization of L-amino acids in proteins in metabolically inert tissues during one's lifetime (Figure 4).

#### Fig. 4. **Significance of D-amino acids in proteins**

D-amino acids in proteins are seen in fossils as well as in the target organs of aging changes and age related disorders such as cataract, Alzheimer's disease, and atherosclerosis.

#### **2.3 Co-localization of advanced glycation end products and D-amino acids in agerelated macular degeneration**

To reveal the role of AGEs and D-amino acids in the development of AMD, we analyzed the immunohistochemical localization of AGE and D-amino acid-containing proteins in human ocular samples of various ages.

Depending on the configuration of the side chains around the chiral carbon atoms, amino acids are either l- or d-amino acids. If amino acids were synthesized chemically, the quantity of l- and d-amino acids would be equal. However, proteins in all living organisms are

L-amino acids are converted to D-amino acids with a half-life of several thousand years, thus creating an equal amount of L- and D-amino acids over a particular period. This amount of D-amino acids is used for the age determination of fossils. Thus, D-amino acids are regarded as the products of post-mortal change and are irrelevant to living organisms. However, biologically uncommon D-amino acids that are enantiomers of L-amino acids have been found in the lenses16-19, teeth20, bones, brains21, skin22, aortas23, erythrocytes24, lungs25, and ligaments of elderly donors.26 The presence of D-amino acids in aged tissues of the living body is considered to be a result of racemization of L-amino acids in proteins in

D-amino acids in proteins are seen in fossils as well as in the target organs of aging changes and age related disorders such as cataract, Alzheimer's disease, and atherosclerosis.

To reveal the role of AGEs and D-amino acids in the development of AMD, we analyzed the immunohistochemical localization of AGE and D-amino acid-containing proteins in human

**2.3 Co-localization of advanced glycation end products and D-amino acids in age-**

composed exclusively of l-amino acids.

metabolically inert tissues during one's lifetime (Figure 4).

Fig. 4. **Significance of D-amino acids in proteins** 

**related macular degeneration** 

ocular samples of various ages.

Eye samples: Nine eyes from 9 donors of 18 to 88 years of age were obtained at the time of necropsy and used as samples for this study. Among them, 4 eyes from donors older than 68 years had drusen.

Antibodies: *N*<sup>ε</sup> -(carboxy) methyl-L-lysine (CML) is the major component of AGEs in the body. A monoclonal antibody to CML was purchased (Transgenic Co. Ltd, Kumamoto, Japan).

The preparation and characterization of the primary antibody of D-β-Asp containing proteins was as described previously.27 The polyclonal antibody to the synthetic peptide called peptide 3R(Gly-Leu-D-β-Asp-Ala-Thy-Gly-Leu-D-β-Asp-Ala-Thy-Gly-Leu-D-β-Asp-Ala-Thy) corresponding to 3 repeats of positions 149–153 of the human α-A-crystallin was prepared and purified.

Immunohistochemistry: Immunohistochemical localization of CML and D-β-Asp containing proteins was investigated using the antibodies mentioned above. After fixation with 10% formalin solution, 4-μm-thick sections of the paraffin-embedded ocular samples were prepared. After deparaffinization, the sections were treated with 2 mg/mL of monoclonal antibody to CML or 1:500 diluted polyclonal antibody to D-β-Asp-containing proteins. After washing with phosphate-buffered saline, the sections were treated with secondary antibodies labeled with a polymer of horse radish peroxidase (Hitofine, Max-PO kit, Nichirei Co. Ltd, Tokyo, Japan). The final products were visualized using diaminobenzidine solution dissolved in phosphate-buffered saline.

No immunoreactivity to CML was seen in the retinas, choroids, or scleras of 5 eyes of donors younger than 18 years of age. In contrast, moderate immunoreactivity was seen in the retinal nerve fiber layers, and strong immunoreactivity was seen in the drusen and the thickened Bruch's membrane of the 5 eyes of donors older than 68 years (Figure 5).

Fig. 5. **Immunohistochemical localization of AGE in human retina and choroid**  No immunoreactivity to CML, a major component of AGEs, is noted in young donor eyes. In contrast, immunoreactivity to CML is seen in the retinal nerve fiber layer (\*), drusen (arrows), and thickened Bruch's membrane (arrowheads).

Non-Enzymatic Post-Translational

Modifications in the Development of Age-Related Macular Degeneration 79

Fig. 7. **Possible mechanism of AMD in relation to AGEs in drusen** 

clinicopathological finding of AMD.

**4. Acknowledgement** 

The constant interaction of RAGE with AGEs in retinal pigment epithelial cells and drusen leads to the expression of VEGF, reactive oxygen species, and the increased expression of RAGE. This process then leads to neovascularization of the retina, which is a typical

Another possible mechanism involves autoantibodies to AGE-modified proteins that have been detected in the elderly or in patients with rheumatic arthritis, which may induce inflammatory changes. Thus, autoimmune reactions may occur in tissues containing AGEs. In fact, proteomic analysis of drusen in human and animal models of AMD has revealed the deposition of IgG and complement factors. In addition, Becerra *et al.* have reported that the pathogenesis of AMD involves inflammatory changes.37 Based on these findings, intravitreal injection of corticosteroids to reduce the inflammation is clinically used to treat AMD.38 At present, the pathological role of D-amino acid containing proteins in the development of AMD is unknown. However, AGEs and D-amino acids need to be targeted for the prevention and treatment of AMD. For example, pyridoxamine inhibits the formation of AGEs in the body, and it has been used in clinical trials for the treatment of diabetic nephropathy. In addition, D-aspartyl endopeptidase has been shown to digest some D-amino acid containing proteins,41 suggesting that an increased expression of the intrinsic D-aspartyl endopeptidase would decrease the amount of D-amino acids-containing proteins in the target organ of the aging process. We suggest that the accumulating data on AGEs

and D-amino acids will pave the way for new therapies for AMD in the near future.

Ministry of Education*,* Culture*,* Sports*,* Science*,* and Technology of Japan.

This work was supported by Grants for Scientific Research 21592216 (2009-2011) from the

Similarly, no immunoreactivity to the D-β-Asp containing proteins was seen in the retinas, choroids, or scleras of donors younger than 18 years. In contrast, strong immunoreactivity was seen in the drusen seen in donors older than 68 years. In addition, moderate immunoreactivity to D-β-Asp containing proteins was seen in the sclera, the internal limiting membrane of retinal vessels, and Bruch membranes (Figure 6).

#### Fig. 6. **Immunohistochemical localization of D-amino acid-containing proteins in human retina and choroid**

No immunoreactivity to D-β-Asp-containing proteins, one of the major components of Damino acids, is noted in young donor eyes. In contrast, the immunoreactivity to D-β-Aspcontaining proteins is seen in the vessel walls (\*), drusen (arrows), and thickened Bruch's membrane (arrowheads).

#### **3. Possible mechanism of age-related macular degeneration**

Drusen, an early sign of AMD, are small lumps of aggregated proteins rich in AGEs and Damino acid containing proteins, which, in addition to being accessory products of the aging process, also possibly accelerate the aging process, suggesting that AGEs and D-amino acids in drusen play a central role in the development of AMD via various mechanisms.

One possible mechanism involves the interaction of AGEs and AGE receptors, which increases inflammation and accelerates neovascularization. AGE-modified proteins are recognized by the receptor for AGE (RAGE),30 galectin-3,31 macrophage scavenger receptors, and CD36.32 Particularly, the interaction of AGEs with RAGE induces inflammatory cytokines such as TNF-α and VEGF.33 RAGEs are expressed on the surface of retinal pigment epithelial cells. Furthermore, the interaction of AGE and RAGE increases the expression of RAGE, which serves as a positive feedback for the reaction. Thus, the constant interaction of AGEs in drusen with RAGE on retinal pigment epithelial cells would increase the expression of VEGF and induce neovascularization, resulting in AMD (Figure 7).

Similarly, no immunoreactivity to the D-β-Asp containing proteins was seen in the retinas, choroids, or scleras of donors younger than 18 years. In contrast, strong immunoreactivity was seen in the drusen seen in donors older than 68 years. In addition, moderate immunoreactivity to D-β-Asp containing proteins was seen in the sclera, the internal limiting

Fig. 6. **Immunohistochemical localization of D-amino acid-containing proteins in human** 

Drusen, an early sign of AMD, are small lumps of aggregated proteins rich in AGEs and Damino acid containing proteins, which, in addition to being accessory products of the aging process, also possibly accelerate the aging process, suggesting that AGEs and D-amino acids

One possible mechanism involves the interaction of AGEs and AGE receptors, which increases inflammation and accelerates neovascularization. AGE-modified proteins are recognized by the receptor for AGE (RAGE),30 galectin-3,31 macrophage scavenger receptors, and CD36.32 Particularly, the interaction of AGEs with RAGE induces inflammatory cytokines such as TNF-α and VEGF.33 RAGEs are expressed on the surface of retinal pigment epithelial cells. Furthermore, the interaction of AGE and RAGE increases the expression of RAGE, which serves as a positive feedback for the reaction. Thus, the constant interaction of AGEs in drusen with RAGE on retinal pigment epithelial cells would increase the expression of VEGF and induce neovascularization, resulting in

in drusen play a central role in the development of AMD via various mechanisms.

No immunoreactivity to D-β-Asp-containing proteins, one of the major components of Damino acids, is noted in young donor eyes. In contrast, the immunoreactivity to D-β-Aspcontaining proteins is seen in the vessel walls (\*), drusen (arrows), and thickened Bruch's

**3. Possible mechanism of age-related macular degeneration** 

membrane of retinal vessels, and Bruch membranes (Figure 6).

**retina and choroid** 

AMD (Figure 7).

membrane (arrowheads).

#### Fig. 7. **Possible mechanism of AMD in relation to AGEs in drusen**

The constant interaction of RAGE with AGEs in retinal pigment epithelial cells and drusen leads to the expression of VEGF, reactive oxygen species, and the increased expression of RAGE. This process then leads to neovascularization of the retina, which is a typical clinicopathological finding of AMD.

Another possible mechanism involves autoantibodies to AGE-modified proteins that have been detected in the elderly or in patients with rheumatic arthritis, which may induce inflammatory changes. Thus, autoimmune reactions may occur in tissues containing AGEs. In fact, proteomic analysis of drusen in human and animal models of AMD has revealed the deposition of IgG and complement factors. In addition, Becerra *et al.* have reported that the pathogenesis of AMD involves inflammatory changes.37 Based on these findings, intravitreal injection of corticosteroids to reduce the inflammation is clinically used to treat AMD.38 At present, the pathological role of D-amino acid containing proteins in the development of AMD is unknown. However, AGEs and D-amino acids need to be targeted for the prevention and treatment of AMD. For example, pyridoxamine inhibits the formation of AGEs in the body, and it has been used in clinical trials for the treatment of diabetic nephropathy. In addition, D-aspartyl endopeptidase has been shown to digest some D-amino acid containing proteins,41 suggesting that an increased expression of the intrinsic D-aspartyl endopeptidase would decrease the amount of D-amino acids-containing proteins in the target organ of the aging process. We suggest that the accumulating data on AGEs and D-amino acids will pave the way for new therapies for AMD in the near future.

#### **4. Acknowledgement**

This work was supported by Grants for Scientific Research 21592216 (2009-2011) from the Ministry of Education*,* Culture*,* Sports*,* Science*,* and Technology of Japan.

Non-Enzymatic Post-Translational

2007;48:3923-3927.

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[20] Masters PM. Stereochemically altered noncollagenous protein from human dentin.

[21] Shapira R, Chou CH. Differential racemization of aspartate and serine in human myelin

[22] Fujii N, Tajima S, Tanaka N, Fujimoto N, Takata T, Shimo-Oka T. The presence of D-

[23] Powell JT, Vine N, Crossman M. On the accumulation of D-aspartate in elastin and

[24] McFadden PN, Clarke S. Methylation at D-aspartyl residues in erythrocytes: possible

[25] Shapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ. Marked longevity of

and nuclear weapons-related radiocarbon. *J Clin Invest* 1991;87:1828-1834. [26] Ritz-Timme S, Laumeier I, Collins M. Age estimation based on aspartic acid racemization in elastin from the yellow ligaments. *Int J Legal Med* 2003;117:96-101. [27] Yang D, Fujii N, Takata T, et al. Immunological detection of D-beta-aspartate-containing

[28] Ishibashi T, Murata T, Hangai M, et al. Advanced glycation end products in age-related

[29] Kaji Y, Oshika T, Takazawa Y, Fukayama M, Fujii N. Accumulation of D-beta-aspartic

[30] Wautier JL, Wautier MP, Schmidt AM, et al. Advanced glycation end products (AGEs)

[32] Ohgami N, Nagai R, Ikemoto M, et al. Cd36, a member of the class b scavenger receptor

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acid-containing proteins in age-related ocular diseases. *Chem Biodivers* 2010;7:1364-

on the surface of diabetic erythrocytes bind to the vessel wall via a specific receptor inducing oxidant stress in the vasculature: a link between surface-associated AGEs

protein for advanced glycation end products (AGE): a new member of the AGE-

family, as a receptor for advanced glycation end products. *J Biol Chem*

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[12] Stitt AW. The maillard reaction in eye diseases. *Ann N Y Acad Sci* 2005;1043:582-597. [13] Kaji Y, Usui T, Ishida S, et al. Inhibition of diabetic leukostasis and blood-retinal barrier

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**5** 

*1,2Brazil 3,4USA* 

**Experimental Treatments for Neovascular** 

*Escola Paulista de Medicina, Universidade Federal de São Paulo, UNIFESP, São Paulo,* 

Age related macular degeneration (AMD) is the leading cause of severe visual loss in adults older than 60 years (1, 2). It is estimated that approximately 30% of adults older than 75 years have some sign of AMD and around 10% develop advanced stages of the disease. More than 1.6 million people in the United States currently have one or both eyes affected by an advanced stage of AMD and it is estimated that there are another 7 million individuals "at risk" (1). Due to rapid aging of the population in many developed countries, this number is expected to double by the year of 2020 (1, 3). Although neovascular AMD only accounts for about 10–20% of the overall AMD incidence, this subtype is responsible

Neovascular AMD is characterized by the presence of choroidal neovascularization (CNV) and is associated with retinal pigment epithelium detachment (PED), retinal pigment epithelium (RPE) tears, fibrovascular disciform scarring, and vitreous hemorrhage(4).

Choroidal neovascularization is an intricate process controlled by myriad angiogenic agents such as growth factors, cytokines, and extracellular matrix (ECM) components. Several growth factors have been implicated in pathologic vessel formation in ocular diseases, such as age-related macular degeneration, including fibroblast growth factor (FGF), plateletderived growth factor (PEDF), tumor necrosis factor (TNF-α) and vascular endothelial growth factor (VEGF)(6). Additionally, it is hypothesized that an inflammatory process is behind the pathogenesis of AMD. It was found that extracellular depositions of diffuse basal laminar and linear deposits (BLD) between the cytoplasmic and basement membrane of the RPE are significantly associated with CNV formation (4, 5, 7). Histological studies of these BLDs proved the presence of complement complexes C3, C5b-9, MMP- 2, MMP-9, and vitronectin (8). Further support of this hypothesis came from genetic studies where

for 90% of cases of severe vision loss (20/200 or worse) (4, 5).

**1. Introduction** 

**Age-Related Macular Degeneration** 

*Harvard Medical School, Department of Ophthalmology, Boston, 4Harvard-MIT Division of Health Sciences and Technology,* 

*Massachusetts Institute of Technology, Cambridge,* 

C. V. Regatieri1,3, J. L. Dreyfuss2,4 and H. B. Nader2

*1Departmento de Oftalmologia, 2Departmento de Bioquímica,* 

*3Schepens Eye Research Institute,* 


### **Experimental Treatments for Neovascular Age-Related Macular Degeneration**

C. V. Regatieri1,3, J. L. Dreyfuss2,4 and H. B. Nader2

*1Departmento de Oftalmologia, 2Departmento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, UNIFESP, São Paulo, 3Schepens Eye Research Institute, Harvard Medical School, Department of Ophthalmology, Boston, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, 1,2Brazil 3,4USA* 

#### **1. Introduction**

82 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

[35] Shibayama R, Araki N, Nagai R, Horiuchi S. Autoantibody against N(epsilon)-

[36] Tai AW, Newkirk MM. An autoantibody targeting glycated IgG is associated with

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[38] Becerra EM, Morescalchi F, Gandolfo F, et al. Clinical evidence of intravitreal

[39] Metz TO, Alderson NL, Thorpe SR, Baynes JW. Pyridoxamine, an inhibitor of advanced

[40] Williams ME, Bolton WK, Khalifah RG, Degenhardt TP, Schotzinger RJ, McGill JB.

type 2 diabetes and overt nephropathy. *Am J Nephrol* 2007;27:605-614. [41] Kinouchi T, Nishio H, Nishiuchi Y, et al. Isolation and characterization of mammalian

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2000;120:188-193.

(carboxymethyl)lysine: an advanced glycation end product of the Maillard reaction.

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triamcinolone acetonide in the management of age-related macular degeneration.

glycation and lipoxidation reactions: a novel therapy for treatment of diabetic

Effects of pyridoxamine in combined phase 2 studies of patients with type 1 and

Age related macular degeneration (AMD) is the leading cause of severe visual loss in adults older than 60 years (1, 2). It is estimated that approximately 30% of adults older than 75 years have some sign of AMD and around 10% develop advanced stages of the disease. More than 1.6 million people in the United States currently have one or both eyes affected by an advanced stage of AMD and it is estimated that there are another 7 million individuals "at risk" (1). Due to rapid aging of the population in many developed countries, this number is expected to double by the year of 2020 (1, 3). Although neovascular AMD only accounts for about 10–20% of the overall AMD incidence, this subtype is responsible for 90% of cases of severe vision loss (20/200 or worse) (4, 5).

Neovascular AMD is characterized by the presence of choroidal neovascularization (CNV) and is associated with retinal pigment epithelium detachment (PED), retinal pigment epithelium (RPE) tears, fibrovascular disciform scarring, and vitreous hemorrhage(4).

Choroidal neovascularization is an intricate process controlled by myriad angiogenic agents such as growth factors, cytokines, and extracellular matrix (ECM) components. Several growth factors have been implicated in pathologic vessel formation in ocular diseases, such as age-related macular degeneration, including fibroblast growth factor (FGF), plateletderived growth factor (PEDF), tumor necrosis factor (TNF-α) and vascular endothelial growth factor (VEGF)(6). Additionally, it is hypothesized that an inflammatory process is behind the pathogenesis of AMD. It was found that extracellular depositions of diffuse basal laminar and linear deposits (BLD) between the cytoplasmic and basement membrane of the RPE are significantly associated with CNV formation (4, 5, 7). Histological studies of these BLDs proved the presence of complement complexes C3, C5b-9, MMP- 2, MMP-9, and vitronectin (8). Further support of this hypothesis came from genetic studies where

AMD Experimental Treatments 85

microscopy showed pathologic evidence of CNV in 60% of the lesions. Frank and coworkers also developed a rat model of CNV in 1989 (18). Also, a diode laser may be used to create the CNV (532 nm, 100 mm 50e100 mW, 0.1 s) and this model has been used to assess aging

Subretinal and/or choroidal neovascularization has been immunologically and mechanically induced in rat and mouse models, primarily by injection of synthetic peptides,

Although there are several transgenic mouse models AMD (22), only a relatively few of the models spontaneously develop CNV. It has become apparent that overexpression of VEGF by the retina or RPE is not enough to elicit CNV in these models and there is a central role of compromised Bruch's membrane in the development of CNV (22). The advantages of these models are the ability to study various biologic components of CNV by comparing with controls and cross breeding experiments. Disadvantages relate to the length of time for the CNV to develop, the relatively small percentages of eyes that develop CNV and the small

Toxicity is a complex event *in vivo*, where there may be direct cellular damage, physiological effects, inflammatory effects and other systemic effects. Currently, it is difficult to monitor systemic and physiological effects *in vitro*, so most assays determine effects at cellular level,

New drugs have to go through extensive cytotoxicity testing before they are released for the use (24, 25). Today there is a continuous search for methods to determine the toxicity by using *in vitro* tests, trying to reduce the number of experiments involving animals (26). Important live-cell functions, including apoptosis, cell adhesion, cell migration and cell proliferation, can be monitored with various *in vitro* tests by using colorimetric and fluorescence assays (27, 28). The most frequently used cell lines are: human retinal pigment epithelial cells (ARPE-19), rat neurosensory retinal cells (R28), rat retinal ganglion cells (RGC-5)(29, 30), the immortalized Muller cell line (MIO-M1) (31) and human umbilical vein

Many of these processes lead to changes in intracellular and membrane components that can be followed with appropriately responsiveness by indicators that could be detected by microscopy, flow cytometry or with a microplate reader. Because cytotoxicity could not be easily defined in terms of a single physiological or morphological parameter, it is often desirable to combine several different measures, such as enzymatic activity, membrane permeability or oxidation–reduction potential. The most common assay to determine the cytotoxicity is the viability assay. The viability is principally used to measure the proportion of viable (life and function) cells after a drug exposure. Most tests verify the cell membrane

endothelial cells (HUVEC) and rabbit aorta endothelial cells (6, 32, 33).

viral vectors containing VEGF, cells and inert synthetic materials (19-21).

**c. Transgenic and knockout mouse models of CNV** 

**3. Retina cytotoxicity assays for new drugs** 

as it relates to CNV formation.

size of the CNV.

**a. "In vitro" assays** 

or cytotoxicity (23).

**b. Surgically induced models of CNV** 

mutations/polymorphisms were found in genes coding for the alternative complement pathway regulator (Factor H and Factor H related proteins) and complement pathway proteins (complement component C2, factor B, and toll-like receptor 4).

Several focal treatments have been proposed and extensively studied to prevent the severe visual loss in neovascular AMD patients including laser photocoagulation (9), photodynamic therapy (PDT) (10) and the combination of PDT with intraocular injections of triamcinolone acetonide. Despite anatomical success, there is a low chance for visual improvement when these treatments are used. In recent years, research has provided new insights into the pathogenesis of macular disease. Today less destructive treatments directly targeting CNV and its pathogenic cascade have become available (8, 11). Antibodies against VEGF uniquely offer a significant chance of increase in visual acuity to patients affected by neovascular AMD.

Currently, inhibition of VEGF-A is the first choice of therapy for neovascular AMD, which not only stabilizes, but also improves visual acuity. The most effective preparations, bevacizumab (Avastin, Genentech Inc, South San Francisco, California) or ranibizumab (Lucentis, Genentech Inc), are recombinant monoclonal antibodies (Fab) that neutralize all biologically active forms of VEGF (12). Two Phase III clinical trials (MARINA and ANCHOR) studied ranibizumab for the treatment of CNV associated with neovascular AMD (13-15). In both of these studies, ranibizumab was administered every 4 weeks (fixed schedule) for up to two years without monthly imaging. Both trials demonstrated prevention of substantial vision loss (lost < 15 letters) in more than 90% of subjects. Additionally, approximately 30% to 40% of the subjects experienced substantial visual acuity gains (gain > 15 letters). Though these dramatic results have revolutionized the treatment of neovascular AMD, the monthly treatment schedule used in the clinical trials has a number of drawbacks including the high number of injections and the lack of efficiency in some patients who do not respond to anti-VEGF therapy (12).

Therefore it is important to continue the study of the CNV physiopathology in order to find new molecules involved in the angiogenesis. In this way it will be possible to develop new drugs to reduce the treatment frequency and to treat patients that don't respond to anti-VEGF therapy.

#### **2. Animal models of choroidal neovascularization**

The development of animal models of CNV has paralleled and contributed to the understanding of the biology of this condition. In addition, these models have also been developed in order to test new treatments.

#### **a. Laser induced models of CNV**

The rst CNV model was developed in primates (16), and coworkers later developed a rat model of CNV in 1989 (17). Those authors created argon laser photocoagulation spots (647 nm, 100 mm, 50e100 mW, 0.1 s) through a dilated pupil with a coverslip over the cornea. The created spots break the Bruch's membrane, with a central bubble formation with or without intraretinal or choroidal hemorrhage. There was uorescein angiographic evidence of CNV in 24% of the created lesions. Examination of enucleated eyes by light and electron

mutations/polymorphisms were found in genes coding for the alternative complement pathway regulator (Factor H and Factor H related proteins) and complement pathway

Several focal treatments have been proposed and extensively studied to prevent the severe visual loss in neovascular AMD patients including laser photocoagulation (9), photodynamic therapy (PDT) (10) and the combination of PDT with intraocular injections of triamcinolone acetonide. Despite anatomical success, there is a low chance for visual improvement when these treatments are used. In recent years, research has provided new insights into the pathogenesis of macular disease. Today less destructive treatments directly targeting CNV and its pathogenic cascade have become available (8, 11). Antibodies against VEGF uniquely offer a significant chance of increase in visual acuity to patients affected by

Currently, inhibition of VEGF-A is the first choice of therapy for neovascular AMD, which not only stabilizes, but also improves visual acuity. The most effective preparations, bevacizumab (Avastin, Genentech Inc, South San Francisco, California) or ranibizumab (Lucentis, Genentech Inc), are recombinant monoclonal antibodies (Fab) that neutralize all biologically active forms of VEGF (12). Two Phase III clinical trials (MARINA and ANCHOR) studied ranibizumab for the treatment of CNV associated with neovascular AMD (13-15). In both of these studies, ranibizumab was administered every 4 weeks (fixed schedule) for up to two years without monthly imaging. Both trials demonstrated prevention of substantial vision loss (lost < 15 letters) in more than 90% of subjects. Additionally, approximately 30% to 40% of the subjects experienced substantial visual acuity gains (gain > 15 letters). Though these dramatic results have revolutionized the treatment of neovascular AMD, the monthly treatment schedule used in the clinical trials has a number of drawbacks including the high number of injections and the lack of

Therefore it is important to continue the study of the CNV physiopathology in order to find new molecules involved in the angiogenesis. In this way it will be possible to develop new drugs to reduce the treatment frequency and to treat patients that don't respond to anti-

The development of animal models of CNV has paralleled and contributed to the understanding of the biology of this condition. In addition, these models have also been

The rst CNV model was developed in primates (16), and coworkers later developed a rat model of CNV in 1989 (17). Those authors created argon laser photocoagulation spots (647 nm, 100 mm, 50e100 mW, 0.1 s) through a dilated pupil with a coverslip over the cornea. The created spots break the Bruch's membrane, with a central bubble formation with or without intraretinal or choroidal hemorrhage. There was uorescein angiographic evidence of CNV in 24% of the created lesions. Examination of enucleated eyes by light and electron

proteins (complement component C2, factor B, and toll-like receptor 4).

efficiency in some patients who do not respond to anti-VEGF therapy (12).

**2. Animal models of choroidal neovascularization** 

developed in order to test new treatments.

**a. Laser induced models of CNV** 

neovascular AMD.

VEGF therapy.

microscopy showed pathologic evidence of CNV in 60% of the lesions. Frank and coworkers also developed a rat model of CNV in 1989 (18). Also, a diode laser may be used to create the CNV (532 nm, 100 mm 50e100 mW, 0.1 s) and this model has been used to assess aging as it relates to CNV formation.

#### **b. Surgically induced models of CNV**

Subretinal and/or choroidal neovascularization has been immunologically and mechanically induced in rat and mouse models, primarily by injection of synthetic peptides, viral vectors containing VEGF, cells and inert synthetic materials (19-21).

#### **c. Transgenic and knockout mouse models of CNV**

Although there are several transgenic mouse models AMD (22), only a relatively few of the models spontaneously develop CNV. It has become apparent that overexpression of VEGF by the retina or RPE is not enough to elicit CNV in these models and there is a central role of compromised Bruch's membrane in the development of CNV (22). The advantages of these models are the ability to study various biologic components of CNV by comparing with controls and cross breeding experiments. Disadvantages relate to the length of time for the CNV to develop, the relatively small percentages of eyes that develop CNV and the small size of the CNV.

#### **3. Retina cytotoxicity assays for new drugs**

#### **a. "In vitro" assays**

Toxicity is a complex event *in vivo*, where there may be direct cellular damage, physiological effects, inflammatory effects and other systemic effects. Currently, it is difficult to monitor systemic and physiological effects *in vitro*, so most assays determine effects at cellular level, or cytotoxicity (23).

New drugs have to go through extensive cytotoxicity testing before they are released for the use (24, 25). Today there is a continuous search for methods to determine the toxicity by using *in vitro* tests, trying to reduce the number of experiments involving animals (26). Important live-cell functions, including apoptosis, cell adhesion, cell migration and cell proliferation, can be monitored with various *in vitro* tests by using colorimetric and fluorescence assays (27, 28). The most frequently used cell lines are: human retinal pigment epithelial cells (ARPE-19), rat neurosensory retinal cells (R28), rat retinal ganglion cells (RGC-5)(29, 30), the immortalized Muller cell line (MIO-M1) (31) and human umbilical vein endothelial cells (HUVEC) and rabbit aorta endothelial cells (6, 32, 33).

Many of these processes lead to changes in intracellular and membrane components that can be followed with appropriately responsiveness by indicators that could be detected by microscopy, flow cytometry or with a microplate reader. Because cytotoxicity could not be easily defined in terms of a single physiological or morphological parameter, it is often desirable to combine several different measures, such as enzymatic activity, membrane permeability or oxidation–reduction potential. The most common assay to determine the cytotoxicity is the viability assay. The viability is principally used to measure the proportion of viable (life and function) cells after a drug exposure. Most tests verify the cell membrane

AMD Experimental Treatments 87

Electrophysiological testing is an effective and objective method to assess the status of the visual pathways. The electroretinogram (ERG) is obtained by recording, through a contact lens electrode on the cornea, the electrical potential generated by the retina in response to a brief stimulus (flash or flicker) of light. ERG is one of the most important examinations for retinal biocompatibility in experimental models, since it is a functional and objective test. In animals, behavioral assessment of visual function is a difficult parameter to be evaluated. Currently, the basis of retinal evaluation for pharmacological and toxicological effects of intravitreally-administered drugs in animals consists of ERG associated with histopathology by light and electron microscopy (44, 45). Toxicity testing can be obtained in rodent as well as non-rodent species for extrapolation to humans for determining risk and safety (46).

Monoclonal antibodies (mAbs) can be used therapeutically due to the binding to molecular targets with high specificity. In ophthalmology, therapeutic mAbs have been introduced recently to treat inflammatory and angiogenic diseases. The rationale for mAb application in ophthalmology also is based on a recent understanding of the molecular biology of various

Recent evidences have shown that the cytokine TNF-α participates actively in the pathogenesis of inflammatory, edematous, neovascular and neurodegenerative ocular, and extra ocular diseases. In addition, the central pathogenic role of TNF in medicine is supported by the clinical efficacy of TNF-α antagonists such as infliximab in randomized controlled trials for various diseases including rheumatoid arthritis (RA) and Crohn's disease (47). Furthermore, although TNF-α is barely detectable in the serum of healthy humans at levels of 10 fg/ml, in patients with systemic inflammatory or neoplastic diseases,

Consecutive studies have described the role of infliximab in the treatment of ocular inflammation. Single or multiple infusions of infliximab at concentrations of 3–10 mg/kg within a 2- to 36-month period have been efficacious in preventing ocular attacks, decreasing relapses, diminishing concomitant corticosteroid use, and controlling disease activity in patients with idiopathic uveitis or uveitis associated with juvenile arthritis,

Regarding ocular neovascularization, one patient with Behcet's disease with uveitis and retinal neovascularization treated with systemic infliximab had regression of new vessels after 8 months. A series of patients receiving 5 mg/kg of infliximab infusions for inflammatory arthritis had remarkable regression of CNV due to AMD (49, 50). The preventive and therapeutic effects of infliximab and etanercept have been studied in a rat model of laser-induced CNV as reported previously by other reports and by our research group (6, 51). In the study by Olson et al., both anti-TNF agents given prophylactically decreased the size and leakage of CNV lesions in these animal models, although in one study only etanercept induced reduction of CNV (52). We performed intravitreal injection of escalating doses of infliximab from 10 to 320 μg in rats after laser-induced CNV. At lower doses, infliximab promoted significant reduction of neovascular complex. However, at

ankylosing spondylitis, Behcet's disease, sarcoidosis, or Crohn's disease (12).

**4. Therapeutic Monoclonal Antibodies** 

**a. Monoclonal Antibody anti-tumor necrosis alpha** 

the levels increase markedly to 50 pg/ml (48).

ocular diseases (12).

integrity by dye exclusion, as Naphtalene Black and Trypan Blue as well as by dye uptake as fluorescein diacetate and propidium iodide (PI) (34, 35). In the first one viable cells are impermeable to the dye, and the analysis is performed by light microscopy. In the second test viable cells uptake diacetyl fluorescein and hydrolyze (esterase) in fluorescein that fluoresce in green, and the nucleus of the non-viable cells are stained by the PI that fluoresce in red, the analysis could be performed both by fluorescence microscopy and flow cytometry (36).

Cell viability also can also be measured by MTT reduction (37) using a microtitration assay in 96 multiwell plates. The reduction of tetrazolium salt (yellow) is reduced in metabolically active cells to form insoluble purple formazan crystals. Other assays include acidotropic stain using acridine orange that concentrates in acidic organelles in a pH-dependent manner. Under fluorescence microscope it is possible to see the metachromatic green or red fluorescence of acridine orange to assess cell viability (38).

Besides viability the apoptosis research is a powerful tool for drug toxicity screening. Apoptosis is the programmed cell death and is characterized morphologically by compaction of the nuclear chromatin, cell-permeability and production of apoptotic bodies. The characteristic observed in apoptotic cells is the fragmentation of the chromatin, degradation of the nuclear envelope and nuclear blebbing, resulting in the formation of micronuclei. A different assay frequently used is the APO-BrdU TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) where DNA strands of apoptotic cells are labeled with BrdUTP, once incorporated into the DNA, BrdU can be detected by an anti‑BrdU antibody conjugated with a enzyme or a fluorescent probe using immunohistochemistry or immunofluorescence (39).

Annexin V is a protein that binds phosphatidylserine located at the cell surface and used to detect apoptotic cells. In apoptotic cells phosphatidylserine is exposed to the outer of the plasma membrane being detected by the annexin V conjugated with a fluorophor. Fluorescent cells could be observed in fluorescence microscope or flow cytometer (33, 40).

#### **b. "In vivo" assays**

Retinal toxicity can be evaluated by intravitreal injections of drugs in rats, mice, rabbits and non-human primates. The safety and efficacy of intravitreal drugs can be analyzed in choroidal neovascularization (CNV) in the laser-induced rat model (6, 41). The investigation of toxicity in animal models using the standard tools of light microscopy (LM) and histopathological analysis makes critical benchmarks for the study of development of the angioproliferative disease. In this way is possible to observe the functional and morphological alteration results of drug toxicity *in vivo*.

Microscopic studies using light, electron or confocal microscopy are common methods used for retinal biocompatibility studies. For microscopy analysis, it is essential to know the normal retina morphology of the animal species analyzed. Histological studies, using light or electron microscopies could be descriptive or analytical.

Clinical evaluation is also an important method to evaluate the retinal toxicity of new drugs. The occurrence of a transient or permanent toxic reaction can be documented by the retinal appearance, function or histological findings in experimental eyes (42). Ocular examinations include slitlamp for anterior segment and detailed dilated fundus examinations (42, 43).

integrity by dye exclusion, as Naphtalene Black and Trypan Blue as well as by dye uptake as fluorescein diacetate and propidium iodide (PI) (34, 35). In the first one viable cells are impermeable to the dye, and the analysis is performed by light microscopy. In the second test viable cells uptake diacetyl fluorescein and hydrolyze (esterase) in fluorescein that fluoresce in green, and the nucleus of the non-viable cells are stained by the PI that fluoresce in red, the

Cell viability also can also be measured by MTT reduction (37) using a microtitration assay in 96 multiwell plates. The reduction of tetrazolium salt (yellow) is reduced in metabolically active cells to form insoluble purple formazan crystals. Other assays include acidotropic stain using acridine orange that concentrates in acidic organelles in a pH-dependent manner. Under fluorescence microscope it is possible to see the metachromatic green or red

Besides viability the apoptosis research is a powerful tool for drug toxicity screening. Apoptosis is the programmed cell death and is characterized morphologically by compaction of the nuclear chromatin, cell-permeability and production of apoptotic bodies. The characteristic observed in apoptotic cells is the fragmentation of the chromatin, degradation of the nuclear envelope and nuclear blebbing, resulting in the formation of micronuclei. A different assay frequently used is the APO-BrdU TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) where DNA strands of apoptotic cells are labeled with BrdUTP, once incorporated into the DNA, BrdU can be detected by an anti‑BrdU antibody conjugated with a enzyme or a fluorescent probe using

Annexin V is a protein that binds phosphatidylserine located at the cell surface and used to detect apoptotic cells. In apoptotic cells phosphatidylserine is exposed to the outer of the plasma membrane being detected by the annexin V conjugated with a fluorophor. Fluorescent cells could be observed in fluorescence microscope or flow cytometer (33, 40).

Retinal toxicity can be evaluated by intravitreal injections of drugs in rats, mice, rabbits and non-human primates. The safety and efficacy of intravitreal drugs can be analyzed in choroidal neovascularization (CNV) in the laser-induced rat model (6, 41). The investigation of toxicity in animal models using the standard tools of light microscopy (LM) and histopathological analysis makes critical benchmarks for the study of development of the angioproliferative disease. In this way is possible to observe the functional and

Microscopic studies using light, electron or confocal microscopy are common methods used for retinal biocompatibility studies. For microscopy analysis, it is essential to know the normal retina morphology of the animal species analyzed. Histological studies, using light

Clinical evaluation is also an important method to evaluate the retinal toxicity of new drugs. The occurrence of a transient or permanent toxic reaction can be documented by the retinal appearance, function or histological findings in experimental eyes (42). Ocular examinations include slitlamp for anterior segment and detailed dilated fundus examinations (42, 43).

analysis could be performed both by fluorescence microscopy and flow cytometry (36).

fluorescence of acridine orange to assess cell viability (38).

immunohistochemistry or immunofluorescence (39).

morphological alteration results of drug toxicity *in vivo*.

or electron microscopies could be descriptive or analytical.

**b. "In vivo" assays** 

Electrophysiological testing is an effective and objective method to assess the status of the visual pathways. The electroretinogram (ERG) is obtained by recording, through a contact lens electrode on the cornea, the electrical potential generated by the retina in response to a brief stimulus (flash or flicker) of light. ERG is one of the most important examinations for retinal biocompatibility in experimental models, since it is a functional and objective test. In animals, behavioral assessment of visual function is a difficult parameter to be evaluated. Currently, the basis of retinal evaluation for pharmacological and toxicological effects of intravitreally-administered drugs in animals consists of ERG associated with histopathology by light and electron microscopy (44, 45). Toxicity testing can be obtained in rodent as well as non-rodent species for extrapolation to humans for determining risk and safety (46).

#### **4. Therapeutic Monoclonal Antibodies**

Monoclonal antibodies (mAbs) can be used therapeutically due to the binding to molecular targets with high specificity. In ophthalmology, therapeutic mAbs have been introduced recently to treat inflammatory and angiogenic diseases. The rationale for mAb application in ophthalmology also is based on a recent understanding of the molecular biology of various ocular diseases (12).

#### **a. Monoclonal Antibody anti-tumor necrosis alpha**

Recent evidences have shown that the cytokine TNF-α participates actively in the pathogenesis of inflammatory, edematous, neovascular and neurodegenerative ocular, and extra ocular diseases. In addition, the central pathogenic role of TNF in medicine is supported by the clinical efficacy of TNF-α antagonists such as infliximab in randomized controlled trials for various diseases including rheumatoid arthritis (RA) and Crohn's disease (47). Furthermore, although TNF-α is barely detectable in the serum of healthy humans at levels of 10 fg/ml, in patients with systemic inflammatory or neoplastic diseases, the levels increase markedly to 50 pg/ml (48).

Consecutive studies have described the role of infliximab in the treatment of ocular inflammation. Single or multiple infusions of infliximab at concentrations of 3–10 mg/kg within a 2- to 36-month period have been efficacious in preventing ocular attacks, decreasing relapses, diminishing concomitant corticosteroid use, and controlling disease activity in patients with idiopathic uveitis or uveitis associated with juvenile arthritis, ankylosing spondylitis, Behcet's disease, sarcoidosis, or Crohn's disease (12).

Regarding ocular neovascularization, one patient with Behcet's disease with uveitis and retinal neovascularization treated with systemic infliximab had regression of new vessels after 8 months. A series of patients receiving 5 mg/kg of infliximab infusions for inflammatory arthritis had remarkable regression of CNV due to AMD (49, 50). The preventive and therapeutic effects of infliximab and etanercept have been studied in a rat model of laser-induced CNV as reported previously by other reports and by our research group (6, 51). In the study by Olson et al., both anti-TNF agents given prophylactically decreased the size and leakage of CNV lesions in these animal models, although in one study only etanercept induced reduction of CNV (52). We performed intravitreal injection of escalating doses of infliximab from 10 to 320 μg in rats after laser-induced CNV. At lower doses, infliximab promoted significant reduction of neovascular complex. However, at

AMD Experimental Treatments 89

An anti-FGF mAb (no registered brand name to date, BioWa, Princeton, NJ, USA) was developed recently for future application on the treatment of various cancers. Although no study has reported if that anti- FGF agent is useful in ocular pharmacology, some potential indications for the application of anti-FGF mAb based on FGF function can be proposed as adjuvant chemotherapy for ocular melanoma, in conjunction with other mAbs such as anti-TNF to treat PVR associated with rhegmatogenous retinal detachment, and to reduce the chance of PCO after cataract surgery (12, 67). More investigation should unravel the usefulness of anti-FGF mAbs in PCO or PVR, because so far the absence of a cause–effect relationship has not been settled. In addition, other mediators may play a more important

Choroidal neovascularization is a complex process controlled by numerous angiogenic agents such as growth factors, cytokines and ECM components, including glycosaminoglycans (GAGs) (68, 69). GAGs can interact with a diverse range of proteins leading to various biological activities, including angiogenesis (69). Among the sulfated GAGs, heparin and heparan sulfate (HS) have been involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions (70-73). This modulation occurs through the interaction of GAGs with angiogenic growth factors, such as VEGF, FGF, TGF-β, IFN-γ and TNF-α. This property of GAGs to bind and modulate angiogenic growth factors provides a strong reason for studying and designing new synthetic GAG analogs, or discovering GAG-like natural compounds, endowed with angiostatic properties. Sulfated oligosaccharides, which are structural mimics of HS or heparin, are potential drug candidates because these compounds may interfere with the role HS plays in the process of angiogenesis. Heparin is known for its anticoagulant activity, but

Recently, we have shown that a heparinoid isolated from marine shrimp presenting negligible anticoagulant and hemorrhagic activities was able to reduce over 60% the neovascularization areas in the laser induced CNV after intravitreal injection. Also this compound is capable of reducing acute inflammatory processes in an animal model (76).

Studies using intravitreal injection of PI88 (phosphomannopentaose sulfate) showed that this compound is capable to reduce the neovascularization area in laser induced experimental CNV in 50% (77). Intravitreal injection of heparin also can reduce the size of

The pharmacological and biochemical properties of the heparinoids point to these

Immunological factors are involved not only in the pathogenesis of AMD, but also in its treatment of this disease. Genetic polymorphisms in different complement proteins can increase the risk for developing AMD (e.g., lack of factor H in patients with Y402H

role than FGF in these entities.

**5. Angiostatic compounds** 

it also has a strong anti-inflammatory effect also (74, 75).

**b. Blockage of complement cascade** 

the CNV, but the hemorrhagic complications are imminent (33, 78).

compounds as compelling drug candidates for treating neovascular AMD.

**a. Heparin mimetics** 

higher doses, it induced no effect compared to the control group. These results suggested that either the pro-angiogenic effect of anti-TNF mAb might occur only at higher doses or that in a lower dose some antiangiogenic indirect effect may be seen. Clinical studies have shown a marked elevation in vitreous levels of TNF-α in patients with PDR (53, 54). Experimental studies in a rat RD (retinal detachment) model showed that anti-TNF agents might reduce leukocyte adhesion, blood–retina barrier breakdown, and endothelial injury. The association between TNF-α and pathologic intraocular neovascularization may be explained by direct transmembrane-TNF stimulation of blood vessel growth, or TNF-αinduced expression of isoform VEGF-C, which may protect retinal endothelial cells from apoptosis (55).

#### **b. Monoclonal Antibody anti-platelet derived growth factor**

Vascular endothelial cells release PDGF-B, which in turn induce recruitment, proliferation, and survival of pericytes, glial cells, and RPE cells (56). Newly established pericytes along with retinal cells provide survival signals for endothelial cells, and more importantly, pericytes may promote the scarring process following CNV (57). Mural cell recruitment to the growing endothelial tube is regulated by PDGF-B signaling; interference with this pathway causes disruption of endothelial cell–mural cell interactions and loss of mural cells. Therefore, antagonists of PDGFs with or without VEGF antagonists may reduce scarring and neovascularization. Moreover, inhibition of both VEGF-A and PDGF-B signaling may be more effective than blocking VEGF-A alone in causing vessel regression in multiple models of neovascular growth (58-60). A clinical trial phase 1 is evaluating the safety of a monoclonal antibody anti PDGF injected intravitreously for the treatment of neovascular AMD (E10030- Ophthotech Corporation, clinical trial NCT00569140) (61).

#### **c. Monoclonal Antibody anti-integrin** α**5**β**1**

Components of the ECM play an important role in angiogenesis and CNV formation by helping to facilitate endothelial cell migration. Integrins are heterodimeric transmembrane proteins, composed of alpha and beta subunits, which interact with the ECM. Both αvβ3 and α5β1 integrins have been shown to play a role in angiogenesis and their expression is upregulated in activated vascular endothelial cells (62). Inhibition of α5β1 integrin may be of particular interest for the treatment of neovascular AMD because of its expression in RPE, macrophages, and fibroblasts in addition to endothelial cells. Wang et al. demonstrated that an integrin α5β1 inhibitor (ATN-161) was able to inhibit CNV leakage and neovascularization in a laser induced CNV model (63).

#### **d. Monoclonal Antibody anti-basic fibroblast growth factor (b-FGF)**

FGFs are a family of heparin-binding growth factors involved in wound healing and embryonic development. The basic-FGF form, also referred to as b-FGF, may be a more potent angiogenic factor than VEGF or PDGF (64). In the eye, FGF is localized within the lacrimal gland, retina, lens, photoreceptors, aqueous humor, vitreous, and corneal epithelium. In both retina and RPE cells, FGF induces changes in cellular proliferation and *in vivo* angiogenesis. Most uveal melanoma cell lines express FGF subtypes including b-FGF to various extents, and increased FGF expression along with other growth factors was reported in an animal model of retinal detachment (65, 66).

higher doses, it induced no effect compared to the control group. These results suggested that either the pro-angiogenic effect of anti-TNF mAb might occur only at higher doses or that in a lower dose some antiangiogenic indirect effect may be seen. Clinical studies have shown a marked elevation in vitreous levels of TNF-α in patients with PDR (53, 54). Experimental studies in a rat RD (retinal detachment) model showed that anti-TNF agents might reduce leukocyte adhesion, blood–retina barrier breakdown, and endothelial injury. The association between TNF-α and pathologic intraocular neovascularization may be explained by direct transmembrane-TNF stimulation of blood vessel growth, or TNF-αinduced expression of isoform VEGF-C, which may protect retinal endothelial cells from

Vascular endothelial cells release PDGF-B, which in turn induce recruitment, proliferation, and survival of pericytes, glial cells, and RPE cells (56). Newly established pericytes along with retinal cells provide survival signals for endothelial cells, and more importantly, pericytes may promote the scarring process following CNV (57). Mural cell recruitment to the growing endothelial tube is regulated by PDGF-B signaling; interference with this pathway causes disruption of endothelial cell–mural cell interactions and loss of mural cells. Therefore, antagonists of PDGFs with or without VEGF antagonists may reduce scarring and neovascularization. Moreover, inhibition of both VEGF-A and PDGF-B signaling may be more effective than blocking VEGF-A alone in causing vessel regression in multiple models of neovascular growth (58-60). A clinical trial phase 1 is evaluating the safety of a monoclonal antibody anti PDGF injected intravitreously for the treatment of neovascular

Components of the ECM play an important role in angiogenesis and CNV formation by helping to facilitate endothelial cell migration. Integrins are heterodimeric transmembrane proteins, composed of alpha and beta subunits, which interact with the ECM. Both αvβ3 and α5β1 integrins have been shown to play a role in angiogenesis and their expression is upregulated in activated vascular endothelial cells (62). Inhibition of α5β1 integrin may be of particular interest for the treatment of neovascular AMD because of its expression in RPE, macrophages, and fibroblasts in addition to endothelial cells. Wang et al. demonstrated that an integrin α5β1 inhibitor (ATN-161) was able to inhibit CNV leakage and

FGFs are a family of heparin-binding growth factors involved in wound healing and embryonic development. The basic-FGF form, also referred to as b-FGF, may be a more potent angiogenic factor than VEGF or PDGF (64). In the eye, FGF is localized within the lacrimal gland, retina, lens, photoreceptors, aqueous humor, vitreous, and corneal epithelium. In both retina and RPE cells, FGF induces changes in cellular proliferation and *in vivo* angiogenesis. Most uveal melanoma cell lines express FGF subtypes including b-FGF to various extents, and increased FGF expression along with other growth factors was

**b. Monoclonal Antibody anti-platelet derived growth factor** 

AMD (E10030- Ophthotech Corporation, clinical trial NCT00569140) (61).

**d. Monoclonal Antibody anti-basic fibroblast growth factor (b-FGF)** 

**c. Monoclonal Antibody anti-integrin** α**5**β**1** 

neovascularization in a laser induced CNV model (63).

reported in an animal model of retinal detachment (65, 66).

apoptosis (55).

An anti-FGF mAb (no registered brand name to date, BioWa, Princeton, NJ, USA) was developed recently for future application on the treatment of various cancers. Although no study has reported if that anti- FGF agent is useful in ocular pharmacology, some potential indications for the application of anti-FGF mAb based on FGF function can be proposed as adjuvant chemotherapy for ocular melanoma, in conjunction with other mAbs such as anti-TNF to treat PVR associated with rhegmatogenous retinal detachment, and to reduce the chance of PCO after cataract surgery (12, 67). More investigation should unravel the usefulness of anti-FGF mAbs in PCO or PVR, because so far the absence of a cause–effect relationship has not been settled. In addition, other mediators may play a more important role than FGF in these entities.

#### **5. Angiostatic compounds**

#### **a. Heparin mimetics**

Choroidal neovascularization is a complex process controlled by numerous angiogenic agents such as growth factors, cytokines and ECM components, including glycosaminoglycans (GAGs) (68, 69). GAGs can interact with a diverse range of proteins leading to various biological activities, including angiogenesis (69). Among the sulfated GAGs, heparin and heparan sulfate (HS) have been involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions (70-73). This modulation occurs through the interaction of GAGs with angiogenic growth factors, such as VEGF, FGF, TGF-β, IFN-γ and TNF-α. This property of GAGs to bind and modulate angiogenic growth factors provides a strong reason for studying and designing new synthetic GAG analogs, or discovering GAG-like natural compounds, endowed with angiostatic properties. Sulfated oligosaccharides, which are structural mimics of HS or heparin, are potential drug candidates because these compounds may interfere with the role HS plays in the process of angiogenesis. Heparin is known for its anticoagulant activity, but it also has a strong anti-inflammatory effect also (74, 75).

Recently, we have shown that a heparinoid isolated from marine shrimp presenting negligible anticoagulant and hemorrhagic activities was able to reduce over 60% the neovascularization areas in the laser induced CNV after intravitreal injection. Also this compound is capable of reducing acute inflammatory processes in an animal model (76).

Studies using intravitreal injection of PI88 (phosphomannopentaose sulfate) showed that this compound is capable to reduce the neovascularization area in laser induced experimental CNV in 50% (77). Intravitreal injection of heparin also can reduce the size of the CNV, but the hemorrhagic complications are imminent (33, 78).

The pharmacological and biochemical properties of the heparinoids point to these compounds as compelling drug candidates for treating neovascular AMD.

#### **b. Blockage of complement cascade**

Immunological factors are involved not only in the pathogenesis of AMD, but also in its treatment of this disease. Genetic polymorphisms in different complement proteins can increase the risk for developing AMD (e.g., lack of factor H in patients with Y402H

AMD Experimental Treatments 91

Gene-based therapy is defined as the introduction, using a vector, of nucleic acids into cells with the objective of changing gene expression to prevent or reverse a pathological process (96). Pro- and antiangiogenic factors regulate the pathogenesis of the ocular neovascularization. Gene transfer to increase expression of endogenous antiangiogenic proteins has the potential to provide long-term stability in patients with AMD (97). There are two routes of administration of viral vectors: intravitreous injection and subretinal injection. The main vectors used for gene transfer are adenovirus, adeno-associated virus

Genes encoding antiangiogenic proteins are genetically inserted in viral vectors. The viral vectors infect animal cells and the overexpression of the antiangiogenic protein can be detected. Pigment epithelium-derived factor (PEDF) is a serine proteinase inhibitor from cultured retinal pigmented epithelial cells, which posses a combination of neurotrophic, antitumoral and antiangiogenic activities. Intravitreous or subretinal injection of adenoviral vector expressing human PEDF suppressed the development of retinal neovascularization (98). In the rat CNV model, the gene transfer of PEDF using ultrasound-mediated

The secreted extracellular domain of VEGF receptor-1, sFlt-1, a soluble form of the Flt-1 VEGF receptor has been used effectively in recombinant adenovirus (Ad)- and recombinant adeno-associated virus (AAV)-mediated antiangiogenic gene therapy to inhibit angiogenesis in CNV animal models (100, 101). The expression of sFlt-1 was associated with the long-term

Endostatin is C-terminal fragment derived from collagen XVIII that inhibits tumor angiogenesis (103). Systemic injection of adenoviral vectors containing a sequence coding for murine endostatin, and the mice injected had the serum levels of endostatin raised up to 10 fold and had nearly complete prevention of CNV (104). Subconjunctival injection of recombinant adeno-associated viral vector expressing human angiostatin reduced alkali

Intravitreal adenovirus-mediated gene transfer of 15-Lipoxygenase-1, an oxidizing enzyme producing reactive lipid hydroperoxides, efficiently inhibited VEGF induced

The treatment of AMD up to 2000 was limited to vessel destructive procedures that did not improve the visual acuity. The development and testing of therapeutic agents that prevent or delay the progression of AMD is urgently needed, from the standpoint of patient care and quality of life, as well as cost savings. The development of new therapies targeting the angiogenic components of CNV could have a significant impact on the health and quality of life of AMD patients. Moreover combination therapy will possibly replace monotherapy as the treatment of choice in order to reduce the frequency of treatment and prevent the late-

**7. Gene therapy** 

(AAV) and lentivirus (96).

microbubbles was able to inhibit effectively the CNV (99).

regression of neovascular vessels in mice and monkey (102).

neovascularization and pathological changes in rabbit eyes (106).

burn-induced corneal angiogenesis (105).

stage complications of neovascular AMD.

**8. Conclusions** 

mutations) (79). There are three pathways of complement activation and all of them activate a final common pathway (C3). Lipofucsin and basal lipid deposits between Bruch's membrane and the retinal pigment epithelium (RPE) cell layer may act as a stimulus for the local activation of the complement system. This may lead to a further growth of the deposits due to the strong chemotactic activity of complement activation products with an influx of inflammatory cells (80). Furthermore these activated RPE cells release angiogenic stimuli leading to choroidal neovascularization (81).

Several agents that modulate different parts of the complement system are in clinical trials. In general, these agents work either by replacing a defective complement component as factor H, that is the central soluble activation inhibitor of the alternative complement pathway, or by blocking the complement pathway C3, the POT-4 (79, 82).

#### **c. Kinase inhibitors**

Another approach to inhibit angiogenic growth factors as VEGF is through inhibition of the downstream signaling pathways targeting the tyrosine kinases. Several inhibitors were tested and a case in point is the intravitreally administered Vatalanib, a VEGF receptor inhibitor that binds to the intracellular kinase domain (83). Other kinases inhibitors currently in development include pazopanib, sorafenib, motesanib, TG100801, as well as AG013736 (84-86).

Sorafenib is an orally active multikinase inhibitor that inhibits the serine/threonine kinases activity of the VEGF receptor. The CNV area in sorafenib-treated rats was significantly reduced in a dose-dependent manner (85). Sorafenib is in phase III trials for renal-cell carcinoma patients.

#### **6. Small interference RNA (siRNA)**

RNA interference is a technology that allows the silencing of genes in animals using therapeutic double-stranded RNA molecules. siRNA molecules induce gene silencing by binding to complementary target RNA molecules in association with the nucleolytic cytoplasmic protein complex known as the RNA-induced silencing complex (87). Nowadays, siRNA is being designed to reduce the production of angiogenic molecules providing potent therapies for ocular neovascularization in patients with AMD. siRNA can be injected into the vitreous cavity or at the subretinal space to treat choroidal neovascularization. This delivery produces local silencing of a gene with small chance for a systemic effect on the same gene (88, 89).

The targeted genes for CNV treatment are mostly VEGF and VEGF receptors (90-92). The silencing of hypoxia inducible factor-1alpha (HIF-1alpha), that regulates the VEGF expression in hypoxic conditions of ocular angiogenesis is also under investigation to treat CNV (93). siRNA targeting the TGF-β, involved in fibrotic scars, seems to be another great potential to treat AMD (94). Furthermore genes associated to photoreceptors degeneration (apoptosis mediators) *c-Jun*, and *Bax* are being tested for futures therapies (95).

A phase I study to investigate the safety, tolerability, pharmacokinetics of a single intravitreous injection of Sirna-027 (siRNA-mediated VEGF silencing) in 26 patients with choroidal neovascularization was completed, and stabilization or improvement in visual acuity and foveal thickness was observed (90).

#### **7. Gene therapy**

90 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

mutations) (79). There are three pathways of complement activation and all of them activate a final common pathway (C3). Lipofucsin and basal lipid deposits between Bruch's membrane and the retinal pigment epithelium (RPE) cell layer may act as a stimulus for the local activation of the complement system. This may lead to a further growth of the deposits due to the strong chemotactic activity of complement activation products with an influx of inflammatory cells (80). Furthermore these activated RPE cells release angiogenic stimuli

Several agents that modulate different parts of the complement system are in clinical trials. In general, these agents work either by replacing a defective complement component as factor H, that is the central soluble activation inhibitor of the alternative complement

Another approach to inhibit angiogenic growth factors as VEGF is through inhibition of the downstream signaling pathways targeting the tyrosine kinases. Several inhibitors were tested and a case in point is the intravitreally administered Vatalanib, a VEGF receptor inhibitor that binds to the intracellular kinase domain (83). Other kinases inhibitors currently in development include pazopanib, sorafenib, motesanib, TG100801, as well as

Sorafenib is an orally active multikinase inhibitor that inhibits the serine/threonine kinases activity of the VEGF receptor. The CNV area in sorafenib-treated rats was significantly reduced in a dose-dependent manner (85). Sorafenib is in phase III trials for renal-cell

RNA interference is a technology that allows the silencing of genes in animals using therapeutic double-stranded RNA molecules. siRNA molecules induce gene silencing by binding to complementary target RNA molecules in association with the nucleolytic cytoplasmic protein complex known as the RNA-induced silencing complex (87). Nowadays, siRNA is being designed to reduce the production of angiogenic molecules providing potent therapies for ocular neovascularization in patients with AMD. siRNA can be injected into the vitreous cavity or at the subretinal space to treat choroidal neovascularization. This delivery produces local silencing of a gene with small chance for a

The targeted genes for CNV treatment are mostly VEGF and VEGF receptors (90-92). The silencing of hypoxia inducible factor-1alpha (HIF-1alpha), that regulates the VEGF expression in hypoxic conditions of ocular angiogenesis is also under investigation to treat CNV (93). siRNA targeting the TGF-β, involved in fibrotic scars, seems to be another great potential to treat AMD (94). Furthermore genes associated to photoreceptors degeneration

A phase I study to investigate the safety, tolerability, pharmacokinetics of a single intravitreous injection of Sirna-027 (siRNA-mediated VEGF silencing) in 26 patients with choroidal neovascularization was completed, and stabilization or improvement in visual

(apoptosis mediators) *c-Jun*, and *Bax* are being tested for futures therapies (95).

pathway, or by blocking the complement pathway C3, the POT-4 (79, 82).

leading to choroidal neovascularization (81).

**c. Kinase inhibitors** 

AG013736 (84-86).

carcinoma patients.

**6. Small interference RNA (siRNA)** 

systemic effect on the same gene (88, 89).

acuity and foveal thickness was observed (90).

Gene-based therapy is defined as the introduction, using a vector, of nucleic acids into cells with the objective of changing gene expression to prevent or reverse a pathological process (96). Pro- and antiangiogenic factors regulate the pathogenesis of the ocular neovascularization. Gene transfer to increase expression of endogenous antiangiogenic proteins has the potential to provide long-term stability in patients with AMD (97). There are two routes of administration of viral vectors: intravitreous injection and subretinal injection. The main vectors used for gene transfer are adenovirus, adeno-associated virus (AAV) and lentivirus (96).

Genes encoding antiangiogenic proteins are genetically inserted in viral vectors. The viral vectors infect animal cells and the overexpression of the antiangiogenic protein can be detected. Pigment epithelium-derived factor (PEDF) is a serine proteinase inhibitor from cultured retinal pigmented epithelial cells, which posses a combination of neurotrophic, antitumoral and antiangiogenic activities. Intravitreous or subretinal injection of adenoviral vector expressing human PEDF suppressed the development of retinal neovascularization (98). In the rat CNV model, the gene transfer of PEDF using ultrasound-mediated microbubbles was able to inhibit effectively the CNV (99).

The secreted extracellular domain of VEGF receptor-1, sFlt-1, a soluble form of the Flt-1 VEGF receptor has been used effectively in recombinant adenovirus (Ad)- and recombinant adeno-associated virus (AAV)-mediated antiangiogenic gene therapy to inhibit angiogenesis in CNV animal models (100, 101). The expression of sFlt-1 was associated with the long-term regression of neovascular vessels in mice and monkey (102).

Endostatin is C-terminal fragment derived from collagen XVIII that inhibits tumor angiogenesis (103). Systemic injection of adenoviral vectors containing a sequence coding for murine endostatin, and the mice injected had the serum levels of endostatin raised up to 10 fold and had nearly complete prevention of CNV (104). Subconjunctival injection of recombinant adeno-associated viral vector expressing human angiostatin reduced alkali burn-induced corneal angiogenesis (105).

Intravitreal adenovirus-mediated gene transfer of 15-Lipoxygenase-1, an oxidizing enzyme producing reactive lipid hydroperoxides, efficiently inhibited VEGF induced neovascularization and pathological changes in rabbit eyes (106).

#### **8. Conclusions**

The treatment of AMD up to 2000 was limited to vessel destructive procedures that did not improve the visual acuity. The development and testing of therapeutic agents that prevent or delay the progression of AMD is urgently needed, from the standpoint of patient care and quality of life, as well as cost savings. The development of new therapies targeting the angiogenic components of CNV could have a significant impact on the health and quality of life of AMD patients. Moreover combination therapy will possibly replace monotherapy as the treatment of choice in order to reduce the frequency of treatment and prevent the latestage complications of neovascular AMD.

AMD Experimental Treatments 93

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**6** 

*Italy* 

**Basic Research and Clinical Application** 

**of Age-Related Macular Degeneration** 

Giuseppe Lo Giudice and Alessandro Galan *San Paolo Ophthalimc Center, San Antonio Hospital* 

**of Drug Delivery Systems for the Treatment** 

The eye is in specific environment to resist pharmaceutical approaches. Vitreoretinal diseases, including age-related macular degeneration (AMD) (Green & Enger, 1993; Sarks, 1976; van der Schaft et al., 1992), are refractory to both topical and systemic pharmacological approaches because of specific environment of the eye. More recently, a variety of pharmacological challenges to treat exudative age-related macular degeneration and macular edema are proceeding into clinical trials, as soon as antivascular endothelial growth factor (anti-VEGF) therapies have been proved to be effective by repeated intravitreal injections. Monthly injections of anti-VEGF therapies to maximize visual potential are a significant treatment burden on the patient. As a result, the need for better treatments for AMD remains (Brown et al., 2006; Geroski & Edelhauser, 2000; Hughes, 2005; Rosenfeld et

**1. Introduction** 

al., 2006) (Figure 1).

Fig. 1. Different methods of drug delivery


### **Basic Research and Clinical Application of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration**

Giuseppe Lo Giudice and Alessandro Galan *San Paolo Ophthalimc Center, San Antonio Hospital Italy* 

#### **1. Introduction**

98 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

[100] Bainbridge JW, Mistry A, De Alwis M, Paleolog E, Baker A, Thrasher AJ, et al.

[101] Lai CM, Brankov M, Zaknich T, Lai YK, Shen WY, Constable IJ, et al. Inhibition of

[102] Lai CM, Shen WY, Brankov M, Lai YK, Barnett NL, Lee SY, et al. Long-term evaluation

[103] Folkman J. Antiangiogenesis in cancer therapy--endostatin and its mechanisms of

[104] Mori K, Ando A, Gehlbach P, Nesbitt D, Takahashi K, Goldsteen D, et al. Inhibition of

[106] Viita H, Kinnunen K, Eriksson E, Lahteenvuo J, Babu M, Kalesnykas G, et al.

expressing secretable endostatin. Am J Pathol. 2001 Jul;159(1):313-20. [105] Cheng HC, Yeh SI, Tsao YP, Kuo PC. Subconjunctival injection of recombinant AAV-

neovascularization. Hum Gene Ther. 2001 Jul 1;12(10):1299-310.

sFlt-1. Gene Ther. 2002 Mar;9(5):320-6.

monkeys. Mol Ther. 2005 Oct;12(4):659-68.

2007;13:2344-52.

Ther. 2009 Dec;20(12):1679-86.

action. Exp Cell Res. 2006 Mar 10;312(5):594-607.

Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor

angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal

of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and

choroidal neovascularization by intravenous injection of adenoviral vectors

angiostatin ameliorates alkali burn induced corneal angiogenesis. Mol Vis.

Intravitreal adenoviral 15-lipoxygenase-1 gene transfer prevents vascular endothelial growth factor A-induced neovascularization in rabbit eyes. Hum Gene The eye is in specific environment to resist pharmaceutical approaches. Vitreoretinal diseases, including age-related macular degeneration (AMD) (Green & Enger, 1993; Sarks, 1976; van der Schaft et al., 1992), are refractory to both topical and systemic pharmacological approaches because of specific environment of the eye. More recently, a variety of pharmacological challenges to treat exudative age-related macular degeneration and macular edema are proceeding into clinical trials, as soon as antivascular endothelial growth factor (anti-VEGF) therapies have been proved to be effective by repeated intravitreal injections. Monthly injections of anti-VEGF therapies to maximize visual potential are a significant treatment burden on the patient. As a result, the need for better treatments for AMD remains (Brown et al., 2006; Geroski & Edelhauser, 2000; Hughes, 2005; Rosenfeld et al., 2006) (Figure 1).

Fig. 1. Different methods of drug delivery

Basic Research and Clinical Application

techniques most suitable for AMD.

**2. Ocular delivery systems** 

**2.1 Ocular barriers** 

(Figure 3)

et al. 2011)

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 101

In the design of a drug delivery system for the eye a balance must be struck between the limitations imposed by the physicochemical properties of the drugs, the limitations imposed by the anatomy and disease state of the eye, and the dosing requirements of the drug for that particular disease. In the recent past years review articles have focused on drug delivery to the eye (Acharya & Young 2004; Andreoli & Miller, 2007; Barar, Javadzadeh & Omidi, 2008; Barocas & Balachandran 2008; Bekeredjian, Katus & Kuecherer, 2006; Booth et al. 2007; Del Amo & Urtti, 2008; Gaudana et al. 2009; Hoffman, 2008; Hsu, 2007; Lee & Robinson, 2009; Lemley & Han, 2007; Liu & Regillo, 2004; Mitra, 2009; Novack, 2009; Sultana et al., 2006). The aim of this chapter is to emphasize recent advances in ocular drug delivery

The eye is in specific environment to resist pharmaceutical approaches (Maurice & Mishima, 1984). Systemically administered drug cannot easily reach the retina and vitreous cavity due to the blood-aqueous barrier, composed of ciliary non-pigmented epithelium and iridal vascular endothelium with tight junction (TJ) and the outer and inner BRB, which are formed by the retinal pigment epithelium (RPE) and retinal vascular endothelium, respectively. On the other hand, an eyeball is covered with collagenous walls (e.g. cornea and sclera) and epithelial and endothelial barriers (e.g. cornea and RPE). These barriers, continuous tear drainage, frontward flow of aqueous humor, and surrounding blood circulations limit the penetration of administered drug (e.g. eye drops and ointments)

Fig. 3. Possible barriers interfering drug delivery into the eye (Tsutomu, Yasuhiko & Hideya

Compliance is also problematic, particularly among patients who have chronic diseases such as glaucoma and refractory chorioretinal diseases, including uveitis, macular edema, neovascular (wet) and atrophic (dry) AMD, and retinitis pigmentosa. In this section, the

function of ocular barrier systems is briefly described (Table 1).

Opthalmic drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist. A significant challenge to the formulator is to circumvent the protective barriers of the eye without causing permanent tissue damage. Development of newer, more sensitive diagnostic techniques and novel therapeutic agents continue to provide ocular delivery systems with high therapeutic efficacy. The goal of pharmacotherapeutics is to treat a disease in a consistent and predictable fashion. The efficacy of a compound is governed by its intrinsic effects on the target site (and any other sites with which it comes into contact), its distribution throughout and its elimination from the body. Alterations to the and elimination of a compound can thus radically alter its efficacy. For regions of the body with a significant barrier to drug permeation, such as the eye and brain, great care should be taken to deliver drugs appropriately (Figure 2).

Fig. 2. Diagram of the brain and spinal cord. Diagram of the brain and spinal cord illustrating how the eye's inner and outer blood-retinal barriers (BRBs) fit into the overall scheme of blood-neural barriers (BNBs) Barriers between the blood and neural tissues are collectively referred to as BNBs and include the blood-brain barrier (A), the blood-CSF barrier (B), the BRB (C), and the blood-spinal cord barrier (D). Considering that several retinal disorders are accompanied by dysfunction or breakdown of this BRB and their associated cell-cell signaling mechanisms, elucidating the nature of the BRB is important for under standing normal health and disease (Choi & Kim, 2008).

In the design of a drug delivery system for the eye a balance must be struck between the limitations imposed by the physicochemical properties of the drugs, the limitations imposed by the anatomy and disease state of the eye, and the dosing requirements of the drug for that particular disease. In the recent past years review articles have focused on drug delivery to the eye (Acharya & Young 2004; Andreoli & Miller, 2007; Barar, Javadzadeh & Omidi, 2008; Barocas & Balachandran 2008; Bekeredjian, Katus & Kuecherer, 2006; Booth et al. 2007; Del Amo & Urtti, 2008; Gaudana et al. 2009; Hoffman, 2008; Hsu, 2007; Lee & Robinson, 2009; Lemley & Han, 2007; Liu & Regillo, 2004; Mitra, 2009; Novack, 2009; Sultana et al., 2006). The aim of this chapter is to emphasize recent advances in ocular drug delivery techniques most suitable for AMD.

### **2. Ocular delivery systems**

#### **2.1 Ocular barriers**

100 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Opthalmic drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist. A significant challenge to the formulator is to circumvent the protective barriers of the eye without causing permanent tissue damage. Development of newer, more sensitive diagnostic techniques and novel therapeutic agents continue to provide ocular delivery systems with high therapeutic efficacy. The goal of pharmacotherapeutics is to treat a disease in a consistent and predictable fashion. The efficacy of a compound is governed by its intrinsic effects on the target site (and any other sites with which it comes into contact), its distribution throughout and its elimination from the body. Alterations to the and elimination of a compound can thus radically alter its efficacy. For regions of the body with a significant barrier to drug permeation, such as the

eye and brain, great care should be taken to deliver drugs appropriately (Figure 2).

Fig. 2. Diagram of the brain and spinal cord. Diagram of the brain and spinal cord illustrating how the eye's inner and outer blood-retinal barriers (BRBs) fit into the overall scheme of blood-neural barriers (BNBs) Barriers between the blood and neural tissues are collectively referred to as BNBs and include the blood-brain barrier (A), the blood-CSF barrier (B), the BRB (C), and the blood-spinal cord barrier (D). Considering that several retinal disorders are accompanied by dysfunction or breakdown of this BRB and their associated cell-cell signaling mechanisms, elucidating the nature of the BRB is important for

under standing normal health and disease (Choi & Kim, 2008).

The eye is in specific environment to resist pharmaceutical approaches (Maurice & Mishima, 1984). Systemically administered drug cannot easily reach the retina and vitreous cavity due to the blood-aqueous barrier, composed of ciliary non-pigmented epithelium and iridal vascular endothelium with tight junction (TJ) and the outer and inner BRB, which are formed by the retinal pigment epithelium (RPE) and retinal vascular endothelium, respectively. On the other hand, an eyeball is covered with collagenous walls (e.g. cornea and sclera) and epithelial and endothelial barriers (e.g. cornea and RPE). These barriers, continuous tear drainage, frontward flow of aqueous humor, and surrounding blood circulations limit the penetration of administered drug (e.g. eye drops and ointments) (Figure 3)

Fig. 3. Possible barriers interfering drug delivery into the eye (Tsutomu, Yasuhiko & Hideya et al. 2011)

Compliance is also problematic, particularly among patients who have chronic diseases such as glaucoma and refractory chorioretinal diseases, including uveitis, macular edema, neovascular (wet) and atrophic (dry) AMD, and retinitis pigmentosa. In this section, the function of ocular barrier systems is briefly described (Table 1).

Basic Research and Clinical Application

**2.3 Choroid/Bruch's membrane and retina** 

drugs.

Bhutto, et al. 2006).

transcellular route (Pederson, 2006).

**2.4 Blood retinal barrier** 

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 103

through blood and lymph (Gausas, Gonnering, Lemke, et al. 1999; Singh, 2003; Sugar, Riazi, & Schaffner, 1957). The conjunctival blood vessels do not form a TJ barrier which means drug molecules can enter into the blood circulation by pinocytosis and/or convective transport through paracellular pores in the vascular endothelial layer. The conjunctival lymphatics act as an efflux system for the efficient elimination from the conjunctival space. Drugs transported by lymphatics in conjunction with the elimination by blood circulation can contribute to systemic exposure, since the interstitial fluid is returned to the systemic

Slera: Scleral permeability has been shown to have a strong dependence on the molecular radius (it consists of collagen fibers and proteoglycans embedded in an extracellular matrix); scleral permeability decreases roughly exponentially with molecular radius (Oyster, 1999). The ideal location for transscleral drug delivery is near the equator at 12–17 mm posterior to the corneoscleral limbus. Hydrophobicity of drugs affects scleral permeability; increase of lipophilicity shows lower permeability; and hydrophilic drugs may diffuse through the aqueous medium of proteoglycans in the fiber matrix pores more easily than lipophilic

Choroid/Bruch's Membrane: Choroid is one of the most highly vascularized tissues of the body. The choroidal capillary endothelial cells are fenestrated and, are relatively large in diameter (20–40 µm). Previous histological studies have shown choroidal thickness changes from 200 µm at birth decreasing to about 80 µm by age 90 (Spaide, 2009). In contrast, Bruch's membrane (BM) causes thickening with age (Tout, Chan-Ling, Hollander, et al. 1993). These changes cause increased calcification of elastic fibers, increased cross-linkage of collagen fibers and increased turnover of glycosaminoglycans. Thickness changes of choroid and BM might affect drug permeability from subconjunctiva or episcleral space into the retina and the vitreous (Farboud, Aotaki-Keen, Miyata, et al. 1999; Handa, Verzijl, Matsunaga, et al. 1999; Hewitt, Nakazawa & Newsome, 1989; Hewitt & Newsome, 1985; Spraul, Lang, Grossniklaus, H.E. et al. 1999; Tout, Chan-Ling, Hollander, et al. 1993; Yamada, Ishibashi,

Retina: in the vitreous drugs are eliminated by two main routes from anterior and posterior segments. Elimination via the posterior route takes place by permeation across the retina. In intact retina, the drugs in the subretinal fluid could either be absorbed by the sensory retinal blood vessels or transported across the RPE, where it may be absorbed into the choroidal vessels or pass through the sclera. Drug transport across the RPE takes place both by transcellular and paracellular routes. The driving forces of outward transport of molecules from the subretinal spaces are hydrostatic and osmotic, and small molecules might transport through the paracellular inter-RPE cellular clefts and by active transport through the

The BRB controls the flux of fluid and blood-borne elements into the neural parenchyma, helping to establish the unique neural environment necessary for proper neural function.

circulation after filtration through lymph nodes (Lee, He, Robinson, et al. 2010).


Table 1. Drug delivery routes for Age-related macular de generation

#### **2.2 Tear, cornea, conjunctiva and sclera**

Tear: One of the precorneal barriers is tear film which reduces the effective concentration of the administrated drugs due to dilution by the tear turnover (approximately 1 µL/min), accelerated clearance, and binding of the drug molecule to the tear proteins (Craig, 2002).

Cornea: The cornea consists of three layers; epithelium, stroma and endothelium, with each one possessing a different polarity, rate-limiting structure for drug penetration and a mechanical barrier to inhibit transport of exogenous substances into the eye (Pederson, 2006). The corneal epithelium is characterized by tight junctions among cells; these are formed to restrict paracellular drug permeation from the tear film. The highly hydrated structure of the stroma (extracellular matrix of a lamellar arrangement of collagen fibrils) acts as a barrier to permeation of lipophilic drug molecules. Corneal endothelium acts as a separating barrier between the stroma and aqueous humor (Fischbarg, 2006). .

Conjunctiva: Conjunctiva or episclera has a rich supply of capillaries and lymphatics therefore, administrated drugs in the conjunctival or episcleral space may be cleared through blood and lymph (Gausas, Gonnering, Lemke, et al. 1999; Singh, 2003; Sugar, Riazi, & Schaffner, 1957). The conjunctival blood vessels do not form a TJ barrier which means drug molecules can enter into the blood circulation by pinocytosis and/or convective transport through paracellular pores in the vascular endothelial layer. The conjunctival lymphatics act as an efflux system for the efficient elimination from the conjunctival space. Drugs transported by lymphatics in conjunction with the elimination by blood circulation can contribute to systemic exposure, since the interstitial fluid is returned to the systemic circulation after filtration through lymph nodes (Lee, He, Robinson, et al. 2010).

Slera: Scleral permeability has been shown to have a strong dependence on the molecular radius (it consists of collagen fibers and proteoglycans embedded in an extracellular matrix); scleral permeability decreases roughly exponentially with molecular radius (Oyster, 1999). The ideal location for transscleral drug delivery is near the equator at 12–17 mm posterior to the corneoscleral limbus. Hydrophobicity of drugs affects scleral permeability; increase of lipophilicity shows lower permeability; and hydrophilic drugs may diffuse through the aqueous medium of proteoglycans in the fiber matrix pores more easily than lipophilic drugs.

#### **2.3 Choroid/Bruch's membrane and retina**

102 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Injection

Direct Transscleral Transchoroidal Transconjunctival/

Minimal injection risk, minimal systemic exposure

80-fold more bioavailable

month 18 Hours to 3 months 30 Minutes to 4 hours <30 Minutes

sub-Tenon's injection

Subconjunctival hemorrhage, suprachoroidal hemorrhage

than

Intravitreal 100% 0.01%–0.1% 0.8%–70% 0%–0.0004% 0%–2%

humor 3% 0.008%–0.8% 0.02%–2.1% 0.0007%–5% 1%–2%

Tear: One of the precorneal barriers is tear film which reduces the effective concentration of the administrated drugs due to dilution by the tear turnover (approximately 1 µL/min), accelerated clearance, and binding of the drug molecule to the tear proteins (Craig, 2002).

Cornea: The cornea consists of three layers; epithelium, stroma and endothelium, with each one possessing a different polarity, rate-limiting structure for drug penetration and a mechanical barrier to inhibit transport of exogenous substances into the eye (Pederson, 2006). The corneal epithelium is characterized by tight junctions among cells; these are formed to restrict paracellular drug permeation from the tear film. The highly hydrated structure of the stroma (extracellular matrix of a lamellar arrangement of collagen fibrils) acts as a barrier to permeation of lipophilic drug molecules. Corneal endothelium acts as a

Conjunctiva: Conjunctiva or episclera has a rich supply of capillaries and lymphatics therefore, administrated drugs in the conjunctival or episcleral space may be cleared

separating barrier between the stroma and aqueous humor (Fischbarg, 2006). .

Suprachoroidal/Intrascleral Hollow Microneedle

Topical Drops Systemic Oral

transscleral Trans-RPE

Safest, but moderate systemic exposure

Conjunctival redness and irritation

Worst bioavailability and second worst duration of action; convenient and can be selfadministered at home

Pills

Minimal local exposure; highest systemic exposure

Gastrointestinal upset

Second worst bioavailability and worst duration of action

Drug Delivery Mode

Safety

Adverse Events

Efficacy

Peak bioavailability

Intra-aqueous

Duration of action

Risk Highest

Pathway to target posterior segment

Intravitreal Injection

injection risk

Most direct and effective (only mode that directly penetrates BRB)

21 Hours to 7 weeks

**2.2 Tear, cornea, conjunctiva and sclera** 

Vitreous hemorrhage, retinal detachment, endophthalmitis Sub-Tenon's Injection

Highest injection risk, mild systemic exposure

Subconjunctival hemorrhage

Much less bioavailable to the vitreous and retina

6 Hours to 1

Table 1. Drug delivery routes for Age-related macular de generation

Choroid/Bruch's Membrane: Choroid is one of the most highly vascularized tissues of the body. The choroidal capillary endothelial cells are fenestrated and, are relatively large in diameter (20–40 µm). Previous histological studies have shown choroidal thickness changes from 200 µm at birth decreasing to about 80 µm by age 90 (Spaide, 2009). In contrast, Bruch's membrane (BM) causes thickening with age (Tout, Chan-Ling, Hollander, et al. 1993). These changes cause increased calcification of elastic fibers, increased cross-linkage of collagen fibers and increased turnover of glycosaminoglycans. Thickness changes of choroid and BM might affect drug permeability from subconjunctiva or episcleral space into the retina and the vitreous (Farboud, Aotaki-Keen, Miyata, et al. 1999; Handa, Verzijl, Matsunaga, et al. 1999; Hewitt, Nakazawa & Newsome, 1989; Hewitt & Newsome, 1985; Spraul, Lang, Grossniklaus, H.E. et al. 1999; Tout, Chan-Ling, Hollander, et al. 1993; Yamada, Ishibashi, Bhutto, et al. 2006).

Retina: in the vitreous drugs are eliminated by two main routes from anterior and posterior segments. Elimination via the posterior route takes place by permeation across the retina. In intact retina, the drugs in the subretinal fluid could either be absorbed by the sensory retinal blood vessels or transported across the RPE, where it may be absorbed into the choroidal vessels or pass through the sclera. Drug transport across the RPE takes place both by transcellular and paracellular routes. The driving forces of outward transport of molecules from the subretinal spaces are hydrostatic and osmotic, and small molecules might transport through the paracellular inter-RPE cellular clefts and by active transport through the transcellular route (Pederson, 2006).

#### **2.4 Blood retinal barrier**

The BRB controls the flux of fluid and blood-borne elements into the neural parenchyma, helping to establish the unique neural environment necessary for proper neural function.

Basic Research and Clinical Application

slows the release.

function of both t and x.

(Knight, 1981)

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 105

diffuse from further and further back within the bulk of the device, which progressively

Systems such as this can be described by solutions to Ficks's second law of diffusion

∂C/∂t = D∂2C/dx2 where C is the concentration in a reservoir, t the time, x the distance and D the diffusion coefficient the diffusant through the media. Partial derivatives (∂) are used because C is a

These formulae can be readily applied to drug release from ointments and gels typically used in eye drops, but an objective difficulty exist in finding a flawless technique to deliver drugs targeting diseases that directly affect the retina and vitreous humor, owing to the anatomic barriers and physiologic clearance mechanisms of the BRB. In this scenario a relevant implication have the importance of the transcellular and paracellular transport pathways across or between epithelial or endothelial BRBs. TJs are the most apical component of both the epithelial or endothelial intercellular junctional complexes. TJs are crucial for the formation and maintenance of epithelial and endothelial paracellular barriers since they semipermeably regulate intercellular passive diffusion of large or hydrophilic ions and solutes. Moreover, a complex network of TJ proteins have been identified, with the assembly and dynamic maintenance of various claudin proteins being the most crucial in regard to dictating the selectivity of this paracellular barrier. Using the transcellular route, drugs are delivered by simple passive diffusion to bind with cell surface membrane–bound transporters (cell surface receptors, pumps, channels, and transporters) where they can directly cross the cell via passive diffusion again or through active transport mechanisms. Various influx and efflux transporters have been found for small lipophilic peptides, organic anions, and cations. Transporter-mediated drug delivery is tissue-specific and has low toxicity since transmembrane concentration gradients are not required for it works. The transcellular pathway is not suitable for high-throughput production of drug candidates though, unlike the paracellular pathway, which is suitable for highthroughput production since drug modification is not needed and one method can be applied for various drugs. Knowing the rate-limiting tissue barrier based on the physiochemical properties of a drug

helps when optimizing the absorption of passively penetrated drugs.

hemorrhage and endophthalmitis (Jager, Aiello, Patel, et al. 2004).

Intraocular drug delivery is the only mode that currently directly broaches the BRB and thereby attains the highest peak intravitreal or intraretinal drug concentrations. However, intraocular drug delivery is the most invasive than other approaches, in that it involves penetrating the globe and thus is not free of injection-related complications. These complications may include trauma and increased risk of cataract, retinal detachment,

Moreover, the presence of some moderate clearance mechanisms (posterior transretinal and anterior aqueous humor elimination pathways) cause the peak drug concentration levels achieved with intraocular administration to decline to non-therapeutic trough levels over

**3.1 Intravitreal injections and intraocular drug delivery** 

BRB restricts drug transport from blood into the retina. BRB is composed of tight junctions (TJ) of retinal capillary endothelial cells and RPE, called iBRB for the inner and oBRB for the outer BRB, respectively. The function of iBRB is supported by Müller cells and astrocytes (Cunha-Vaz, 1979).

Müller cells are known to support neuronal activity and maintain the proper functioning of the iBRB under normal conditions.The astrocytes are closely associated with the retinal capillary vessels and help to maintain the capillary integrity (Zhang & Stone, 1997). Astrocytes are known to increase the barrier properties of the retinal vascular endothelium by enhancing the expression of the tight junction protein ZO-1 and modifying endothelial morphology (Gardner, Lieth, Khin, et al. 1997).

Following systemic drug administration, drugs can easily enter into the choroid since choroid is highly vascularized tissue compared to retina. The choriocapillaris are fenestrated resulting in rapid equilibration of drug molecules present in the bloodstream with the extravascular space of the choroid. Therefore, oBRB (RPE) restricts further entry of drugs from the choroid into the retina. RPE is a monolayer of highly specialized hexagonal-shaped cells, located between the sensory retina and the choroid. The TJ of the RPE efficiently restrict intercellular permeation into the sensory retina. Dysfunction of Müller cells may contribute to a breakdown of the iBRB in many pathological conditions (Tretiach, Madigan, Wen, et al. 2005). Müller cells enhance the secretion of VEGF under hypoxic and inflammatory conditions (Eichler, Kuhrt, Hoffmann, et al. 2000; Kaur, Sivakumar & Foulds, 2006). VEGF-induced occluding phosphorylation and ubiquitination causes trafficking of tight junction and leads to increased retinal vascular permeability (Murakami, Felinski & Antonetti, 2009).

#### **3. Drug delivery systems to posterior segment of the eye and specific delivery systems**

In drug delivery, a more common situation is one where the rate of change of concentration is directly proportional to the concentration of drug present, and drug's potential to diffuse from one region to another is directly proportional to its chemical potential, which can usually be approximated to its concentration. This situation is termed first order and can be expressed as (Higuchi, 1961)

$$-\text{d}\mathbf{c}/\mathbf{dt} = \mathbf{k}\mathbf{c}$$

where dc is the change in concentration, dt is the time interval over which that change occurs, k is a constant, also known as the rate constant, and it is in function of concentration.

Integrating this with respect to time gives (Peppas, 1995)

$$\ln\text{C}\_t - \ln\text{C}\_0 = \text{-kt}$$

where C0 is the initial concentration and Ct is the concentration at time t.

A more common situation arises when drug release is both a function of its concentration within a vehicle and its ability to diffusion through it. When placed into a release medium, the drug closest to the surface is released the fastest. Over a period of time, the drug must

BRB restricts drug transport from blood into the retina. BRB is composed of tight junctions (TJ) of retinal capillary endothelial cells and RPE, called iBRB for the inner and oBRB for the outer BRB, respectively. The function of iBRB is supported by Müller cells and astrocytes

Müller cells are known to support neuronal activity and maintain the proper functioning of the iBRB under normal conditions.The astrocytes are closely associated with the retinal capillary vessels and help to maintain the capillary integrity (Zhang & Stone, 1997). Astrocytes are known to increase the barrier properties of the retinal vascular endothelium by enhancing the expression of the tight junction protein ZO-1 and modifying endothelial

Following systemic drug administration, drugs can easily enter into the choroid since choroid is highly vascularized tissue compared to retina. The choriocapillaris are fenestrated resulting in rapid equilibration of drug molecules present in the bloodstream with the extravascular space of the choroid. Therefore, oBRB (RPE) restricts further entry of drugs from the choroid into the retina. RPE is a monolayer of highly specialized hexagonal-shaped cells, located between the sensory retina and the choroid. The TJ of the RPE efficiently restrict intercellular permeation into the sensory retina. Dysfunction of Müller cells may contribute to a breakdown of the iBRB in many pathological conditions (Tretiach, Madigan, Wen, et al. 2005). Müller cells enhance the secretion of VEGF under hypoxic and inflammatory conditions (Eichler, Kuhrt, Hoffmann, et al. 2000; Kaur, Sivakumar & Foulds, 2006). VEGF-induced occluding phosphorylation and ubiquitination causes trafficking of tight junction and leads to increased retinal vascular permeability (Murakami, Felinski &

**3. Drug delivery systems to posterior segment of the eye and specific** 

In drug delivery, a more common situation is one where the rate of change of concentration is directly proportional to the concentration of drug present, and drug's potential to diffuse from one region to another is directly proportional to its chemical potential, which can usually be approximated to its concentration. This situation is termed first order and can be


ln Ct – ln C0 = - kt

A more common situation arises when drug release is both a function of its concentration within a vehicle and its ability to diffusion through it. When placed into a release medium, the drug closest to the surface is released the fastest. Over a period of time, the drug must

(Cunha-Vaz, 1979).

Antonetti, 2009).

**delivery systems** 

expressed as (Higuchi, 1961)

Integrating this with respect to time gives (Peppas, 1995)

where C0 is the initial concentration and Ct is the concentration at time t.

morphology (Gardner, Lieth, Khin, et al. 1997).

diffuse from further and further back within the bulk of the device, which progressively slows the release.

Systems such as this can be described by solutions to Ficks's second law of diffusion (Knight, 1981)

$$
\partial \mathbf{C} / \partial \mathbf{t} = \mathbf{D} \partial^2 \mathbf{C} / \mathbf{d} \mathbf{x}^2
$$

where C is the concentration in a reservoir, t the time, x the distance and D the diffusion coefficient the diffusant through the media. Partial derivatives (∂) are used because C is a function of both t and x.

These formulae can be readily applied to drug release from ointments and gels typically used in eye drops, but an objective difficulty exist in finding a flawless technique to deliver drugs targeting diseases that directly affect the retina and vitreous humor, owing to the anatomic barriers and physiologic clearance mechanisms of the BRB. In this scenario a relevant implication have the importance of the transcellular and paracellular transport pathways across or between epithelial or endothelial BRBs. TJs are the most apical component of both the epithelial or endothelial intercellular junctional complexes. TJs are crucial for the formation and maintenance of epithelial and endothelial paracellular barriers since they semipermeably regulate intercellular passive diffusion of large or hydrophilic ions and solutes. Moreover, a complex network of TJ proteins have been identified, with the assembly and dynamic maintenance of various claudin proteins being the most crucial in regard to dictating the selectivity of this paracellular barrier. Using the transcellular route, drugs are delivered by simple passive diffusion to bind with cell surface membrane–bound transporters (cell surface receptors, pumps, channels, and transporters) where they can directly cross the cell via passive diffusion again or through active transport mechanisms. Various influx and efflux transporters have been found for small lipophilic peptides, organic anions, and cations. Transporter-mediated drug delivery is tissue-specific and has low toxicity since transmembrane concentration gradients are not required for it works. The transcellular pathway is not suitable for high-throughput production of drug candidates though, unlike the paracellular pathway, which is suitable for highthroughput production since drug modification is not needed and one method can be applied for various drugs. Knowing the rate-limiting tissue barrier based on the physiochemical properties of a drug helps when optimizing the absorption of passively penetrated drugs.

#### **3.1 Intravitreal injections and intraocular drug delivery**

Intraocular drug delivery is the only mode that currently directly broaches the BRB and thereby attains the highest peak intravitreal or intraretinal drug concentrations. However, intraocular drug delivery is the most invasive than other approaches, in that it involves penetrating the globe and thus is not free of injection-related complications. These complications may include trauma and increased risk of cataract, retinal detachment, hemorrhage and endophthalmitis (Jager, Aiello, Patel, et al. 2004).

Moreover, the presence of some moderate clearance mechanisms (posterior transretinal and anterior aqueous humor elimination pathways) cause the peak drug concentration levels achieved with intraocular administration to decline to non-therapeutic trough levels over

Basic Research and Clinical Application

or through the use of a sustained release delivery device.

**3.2 Non-biodegradable, biodegradable implants in AMD** 

acid) (PLGA), poly(caprolactone) and poly(methylene malonate) (table 2).

acetonide illuvien ® IVT, implant Polyimide/PVA wet AMD, DME Brimonidina - IVT, implant PLGA Dry AMD, RP

The ongoing clinical trial clearly indicates that biodegradable polymers are biocompatible. In the near future, many types of biodegradable implants and microparticles will proceed

An injectable, rod-shaped intravitreal implant with FA (Iluvien®, Alimera Sciences, Alpharetta, GA, U.S.) (length: 3.5 mm, diameter: 0.37 mm) has been developed for the treatment of diabetic macular edema (DME). Iluvien® is made of polyimide and PVA, small enough to be injected using an inserter with a 25-gauge needle and is expected to provide sustained delivery of FA to the back of the eye for up to three years. In addition to DME, Iluvien® is in Phase II for the treatment of wet AMD compared to Lucentis®

excipient target indication

membrane/ARPE-19 RP, dry AMD

Active ingredient Brand name Dosage form Release-controlling

(NT-501) - IVT, implant semipermeabile

Table 2. Current and future drugs in clinical trials for posterior disease (AMD)

fluocinolone

for clinical trial.

CNTF

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 107

may act through formulation modification to decrease the solubility of the drug via a suspension or to enhance the residence time in vitreous humor via a biodegradable implant,

Non-biodegradable implants are a reservoir type, which possesses a coating of nonbiodegradable polymers such as poly (vinyl alcohol) (PVA), ethylene vinyl acetate (EVA), and silicone laminate, reserving drug in the inner space. This type exhibits the most stable and long-standing release profile of drug, as compared with other types of implants, because it can reserve a large amount of drug and regulate drug release just by surface area and thickness of PVA, a permeable polymer (Okabe, Kimura, Okabe, et al. 2003; Yasukawa, Ogura, Kimura, et al. 2006; Yasukawa, Ogura, Tabata, et al. 2004). However should be considered the weakness of this type of device involving relatively large size of the device requiring a large incision for implantation, which may increase the risk of retinal complications (vitreous hemorrhage, epiretinal membrane, and retinal detachment), and potential need of removal surgery to exchange the implant or treat possible complications such as retinal detachment and drug-induced adverse effects (Callanan, 2007). As compared with non-biodegradable implants, biodegradable implants have following merits: no need of removal surgery and the flexibility in shape. They can be processed into a variety of configurations involving microparticles, a rod, a disc, or tablet. Thus, the release profile of drug from this 2nd generation biodegradable implants may become as stable as nonbiodegradable ones, while the duration of drug release may be shorter due to the limitation of drug contained. The drugs in the biodegradable implants are conjugated to a variety of polymers including poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic

time, unless the injections are given frequently and repeatedly. The disadvantage caused by the short to medium duration of action of intravitreal drug solutions has been partially alleviated through product formulation or drug device development. Currently, despite its shortcomings, intravitreal administration is the preferred drug delivery route to treat diseases of the posterior segment being the better approach achieving the highest intraocular bioavailability in posterior segment tissues such as the cone-containing macula or fovea.

Anti-VEGF drugs, such as pegaptanib, ranibizumab and bevacizumab are new intravitreal treatments for AMD. To understand the pharmacokinetics of intravitreal drug delivery a great attention should be made to understanding the molecular structure of the normal human vitreous gel and that normal vitreous gel undergoes age-related degenerative liquefaction. It was shown that immediately after intravitreal injection, drugs initially concentrate near the injection site or in the cisternae forming vitreous concentration gradients before distributing throughout the entire vitreous cavity and then reaching steady state equilibrium levels (Lee SS, et al. IOVS 2009;50:ARVO E-Abstract 5950). The location of implant placement in the vitreous (anterior placement behind lens versus posterior placement near the retina) influences drug levels in various tissues. When the implant was placed in the posterior region, drug levels were higher in the posterior retina, choroid-RPE and sclera and lower in the anterior retina, choroid-RPE, and sclera. Posterior placement of the implant is likely to support release of the drug to target tissues in case of retinal disorders. Although the posterior implant reduced drug levels in the lens, no such advantage was found with respect to corticosteroid levels in the trabecular meshwork, possibly due to the high affinity of the drug for this tissue.

After intravitreal placement of an implant, drug levels are not likely to achieve a uniform level throughout the vitreous. However, it is probable that a slow release system achieves zones of steady state concentration, with the contours dictated by the clearance mechanisms of a given therapeutic agent. The reason for such zones or contours of steady state concentration is not necessarily diffusion limitation, but rather the presence of continuous dynamic clearance mechanisms in the posterior segment. There are two main mechanisms of drug clearance for intravitreally administered drugs in the eye: the anterior elimination pathway via counterdirectional bulk aqueous flow and the posterior elimination pathway via vitreoretinochoroidal bulk flow due to hydrostatic and osmotic pressure gradients in the inner, middle, and outer coats of the posterior segment (Kim, Lutz, Wang, et al. 2007). An additional mechanism to consider is the transcellular carrier–mediated transporters found on the RPE. Influx transporters enhance the penetration of drugs and efflux transporters inhibit retention of drugs across the outer (o)BRB (Mannermaa, Vellonen & Urtti, 2006). The duration of action of an intravitreally administered drug may in part depend on the retention of the injected drug at the site of administration. The longer the intravitreal halflife, the greater the anticipated duration of therapeutic response. The half-lives of drugs that are eliminated through both the retina and the aqueous humor, such as small lipophilic drugs, tend to be shorter than the half-lives of drugs eliminated primarily via the anterior route, such as large hydrophilic drugs, and also intravitreal drug elimination depends on the molecular weight (MW) of the drug, with larger MW (> 70,000) drugs displaying longer half-lives. To overcoming the short to medium duration of action of intravitreal drug solutions is the use of one of several available sustained drug release systems. These systems may act through formulation modification to decrease the solubility of the drug via a suspension or to enhance the residence time in vitreous humor via a biodegradable implant, or through the use of a sustained release delivery device.

#### **3.2 Non-biodegradable, biodegradable implants in AMD**

106 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

time, unless the injections are given frequently and repeatedly. The disadvantage caused by the short to medium duration of action of intravitreal drug solutions has been partially alleviated through product formulation or drug device development. Currently, despite its shortcomings, intravitreal administration is the preferred drug delivery route to treat diseases of the posterior segment being the better approach achieving the highest intraocular bioavailability in posterior segment tissues such as the cone-containing macula

Anti-VEGF drugs, such as pegaptanib, ranibizumab and bevacizumab are new intravitreal treatments for AMD. To understand the pharmacokinetics of intravitreal drug delivery a great attention should be made to understanding the molecular structure of the normal human vitreous gel and that normal vitreous gel undergoes age-related degenerative liquefaction. It was shown that immediately after intravitreal injection, drugs initially concentrate near the injection site or in the cisternae forming vitreous concentration gradients before distributing throughout the entire vitreous cavity and then reaching steady state equilibrium levels (Lee SS, et al. IOVS 2009;50:ARVO E-Abstract 5950). The location of implant placement in the vitreous (anterior placement behind lens versus posterior placement near the retina) influences drug levels in various tissues. When the implant was placed in the posterior region, drug levels were higher in the posterior retina, choroid-RPE and sclera and lower in the anterior retina, choroid-RPE, and sclera. Posterior placement of the implant is likely to support release of the drug to target tissues in case of retinal disorders. Although the posterior implant reduced drug levels in the lens, no such advantage was found with respect to corticosteroid levels in the trabecular meshwork,

After intravitreal placement of an implant, drug levels are not likely to achieve a uniform level throughout the vitreous. However, it is probable that a slow release system achieves zones of steady state concentration, with the contours dictated by the clearance mechanisms of a given therapeutic agent. The reason for such zones or contours of steady state concentration is not necessarily diffusion limitation, but rather the presence of continuous dynamic clearance mechanisms in the posterior segment. There are two main mechanisms of drug clearance for intravitreally administered drugs in the eye: the anterior elimination pathway via counterdirectional bulk aqueous flow and the posterior elimination pathway via vitreoretinochoroidal bulk flow due to hydrostatic and osmotic pressure gradients in the inner, middle, and outer coats of the posterior segment (Kim, Lutz, Wang, et al. 2007). An additional mechanism to consider is the transcellular carrier–mediated transporters found on the RPE. Influx transporters enhance the penetration of drugs and efflux transporters inhibit retention of drugs across the outer (o)BRB (Mannermaa, Vellonen & Urtti, 2006). The duration of action of an intravitreally administered drug may in part depend on the retention of the injected drug at the site of administration. The longer the intravitreal halflife, the greater the anticipated duration of therapeutic response. The half-lives of drugs that are eliminated through both the retina and the aqueous humor, such as small lipophilic drugs, tend to be shorter than the half-lives of drugs eliminated primarily via the anterior route, such as large hydrophilic drugs, and also intravitreal drug elimination depends on the molecular weight (MW) of the drug, with larger MW (> 70,000) drugs displaying longer half-lives. To overcoming the short to medium duration of action of intravitreal drug solutions is the use of one of several available sustained drug release systems. These systems

possibly due to the high affinity of the drug for this tissue.

or fovea.

Non-biodegradable implants are a reservoir type, which possesses a coating of nonbiodegradable polymers such as poly (vinyl alcohol) (PVA), ethylene vinyl acetate (EVA), and silicone laminate, reserving drug in the inner space. This type exhibits the most stable and long-standing release profile of drug, as compared with other types of implants, because it can reserve a large amount of drug and regulate drug release just by surface area and thickness of PVA, a permeable polymer (Okabe, Kimura, Okabe, et al. 2003; Yasukawa, Ogura, Kimura, et al. 2006; Yasukawa, Ogura, Tabata, et al. 2004). However should be considered the weakness of this type of device involving relatively large size of the device requiring a large incision for implantation, which may increase the risk of retinal complications (vitreous hemorrhage, epiretinal membrane, and retinal detachment), and potential need of removal surgery to exchange the implant or treat possible complications such as retinal detachment and drug-induced adverse effects (Callanan, 2007). As compared with non-biodegradable implants, biodegradable implants have following merits: no need of removal surgery and the flexibility in shape. They can be processed into a variety of configurations involving microparticles, a rod, a disc, or tablet. Thus, the release profile of drug from this 2nd generation biodegradable implants may become as stable as nonbiodegradable ones, while the duration of drug release may be shorter due to the limitation of drug contained. The drugs in the biodegradable implants are conjugated to a variety of polymers including poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) and poly(methylene malonate) (table 2).


Table 2. Current and future drugs in clinical trials for posterior disease (AMD)

The ongoing clinical trial clearly indicates that biodegradable polymers are biocompatible. In the near future, many types of biodegradable implants and microparticles will proceed for clinical trial.

An injectable, rod-shaped intravitreal implant with FA (Iluvien®, Alimera Sciences, Alpharetta, GA, U.S.) (length: 3.5 mm, diameter: 0.37 mm) has been developed for the treatment of diabetic macular edema (DME). Iluvien® is made of polyimide and PVA, small enough to be injected using an inserter with a 25-gauge needle and is expected to provide sustained delivery of FA to the back of the eye for up to three years. In addition to DME, Iluvien® is in Phase II for the treatment of wet AMD compared to Lucentis®

Basic Research and Clinical Application

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 109

WO2003070918 Technology of siRNA modification to improve biological stability WO2004015107 Technology of siRNA modification to improve biological stability US20100015158 Intraocular delivery of anti-angiogenic antibodies in a liquid or solid polymeric vehicle such as hyaluronic acid or PLGA WO2010009034 Intraocular delivery of anti-angiogenic antibodies in a liquid or solid polymeric vehicle such as hyaluronic acid or PLGA US20100098772 Delivery of anti-angiogenic agents with polymeric hyaluronic acid US20100074957 Injectable intraocular drug delivery system by use of microspheres

US20090258924 Intraocular delivery of siRNA with a polymeric component

Periocular delivery includes such avenues as subconjunctival, retrobulbar, peribulbar, sub-Tenon's and intrascleral delivery. This route requires drugs to pass through several layers of ocular tissue (episclera, sclera, choroid, Bruch's membrane, and RPE) to reach the retina or vitreous humor. Current knowledge shows that the combined effects of several static anatomic barriers and dynamic clearance mechanisms generally make periocular drug delivery one of the least effective ways of attaining high peak therapeutic intraocular drug concentrations in the retina or vitreous. These barriers are categorized into three major groups: static, dynamic and metabolic. Static barriers include the tissues that must be penetrated (e.g., sclera, Bruch's membrane-choroid and RPE). Dynamic barriers include blood flow, lymphatic drainage, transport proteins of the RPE, drug efflux pumps, organic ion transporters and bulk fluid flow from intraocular drainage systems. Metabolic barriers include enzyme systems such as cytochrome P450 and lysosomal enzymes, which have the ability to degrade or detoxify drugs. In addition to the static, dynamic and metabolic barriers, other factors must be considered in periocular delivery such the individual pharmacokinetic properties of the drug (molecular dimensions, molecular weight, atomic charge and chemical components of the drug). Taken together these factors result in low intraocular bioavailability of drug delivered by the various periocular drug techniques,

Overall, when the safety and efficacy of this drug delivery route is compared with that of the others for the treatment of disease in the inner coat of the posterior segment, periocular injection is one of the best for safety, and it is in the middle to low end for efficacy, after intrinsic drug properties are factored in, such as potency and exposure–response relationships. With a few modifications, this route has the greatest potential to surpass intravitreal injection as the preferred treatment option for disease of the inner coat of the eye, since it deposits drug locally immediately adjacent to the targeted tissue without being

A major adjuvant used to overcome the short to medium duration of action of periocular drug solutions is the development of several sustained drug release systems, whether through formulation modifications or through various sustained release drug delivery devices. Many of these options include liposomes, microspheres, and microcapsules with

Patent No. Activity

Table 3. List of Patents on Intraocular Drug Delivery Systems

**3.3 Periocular dug derlivery route in AMD** 

compared with that of intravitreal injection.

overly invasive.

(ClinicalTrials.gov The MAP Study: Fluocinolone acetonide (FA)/MedidurTM for age related macular degeneration (AMD) Pilot. (2010). Available online: http://clinicaltrials.gov/ct2/show/ NCT00605423?term=Iluvien&rank=12), dry AMD (ClinicalTrials.gov Fluocinolone acetonide intravitreal inserts in geographic atrophy. (2010). Available online: http://clinicaltrials.gov/ct2/show/NCT00695318?term=Iluvien&rank=15.

Brimonidine is an a2 adrenergic agonist, which can release various neurotrophins including brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) (Lonngren, Napankangas, Lafuente, et al. 2006; Kim, Chang, Kim, et al. 2007). These neurotrophins have a potential to prevent apoptosis of photoreceptors and/or RPE (Azadi, Johnson, Paquet-Durand, et al. 2007; Zhang, Mo, Fang, et al. 2009). PLGA intravitreal implant with two doses (200 mg, 400 mg) of brimonidine tartrate similar to Ozurdex® is now in Phase II clinical study for dry AMD (ClinicalTrials.gov Safety and efficacy of brimonidine intravitreal implant in patients with geographic atrophy due to age-related macular degeneration (AMD). (2010). Available online: http://clinicaltrials.gov/ct2/show/NCT00658619), and phase I/II clinical trials for RP (ClinicalTrials.gov An exploratory study to evaluate the safety of brimonidine intravitreal implant in patients with retinitis pigmentosa. (2010). Available online: http://clinicaltrials.gov/ct2/show/ NCT00661479) by Allergan, Inc. Neurotech Pharmaceuticals, Inc. (Lincoln, RI, U.S.) has been developing ―"Encapsulated Cell Technology", this is an implantable intravitreal device that uses genetically modified ARPE-19 RPE cells (transfected with plasmid gene insertion techniques) to release a therapeutic growth factor protein, ciliary neurotrophic factor (CNTF), with zero-order kinetics. CNTF is a neurotrophic growth factor with profound antiapoptotic effects that prolong the lives of dying photoreceptor cells. The cells are encapsulated within a semipermeable, hollow-fiber membrane, intravitreal implant device so that the CNTF protein is released to ocular tissues but the modified RPE cells are immunologically isolated from the patient, avoiding immune rejection. Phase 2 trials showed that this delivery system was safe, and a dose-dependent biological effect on the retina was observed. Potential visual benefit was also shown in patients with geographic atrophy (US National Institutes of Health. ClinicalTrials.org. NCT00447954 CgI. A Study of an Encapsulated Cell Technology (ECT) Implant for Patients With Atrophic Macular Degeneration. Phase II study. (2009). http://clinicaltrials.gov/ct2/show/NCT00447954). The duration of action of this encapsulated cell therapy device was found to be up to 2 years. Next-phase clinical trials are under way to continue to evaluate encapsulated cell technology (ECT)-CNTF's effectiveness for the treatment of dry AMD and retinitis pigmentosa.

Anti-VEGF compounds are now required to be administered into the eye every 4 to 6 weeks. Moreover, vitrectomized eyes highly increase the clearance rate of injected drugs and are, therefore, refractory to these drugs. The companies are currently interested in new methods of prolongation of residence time of intravitreally-injected drugs such as ranibizumab and pegaptanib. Viscous medium such as hyaluronic acid or gelling solution are considered as an additive to prolong drug residence in the vitreous cavity (Table 3) (Lyons, Ma & Trogde, 2009; Giese, Kaufmann & Klippel-Giese, 2004; McSwiggen, Beigelman, Macejak, et al. 2003; Robinson, Blanda, Hughes, et al. 2010; Robinson, Blanda, Liu, et al. 2010; Robinson, Tsai, Almazan, et al. 2010).


Table 3. List of Patents on Intraocular Drug Delivery Systems

#### **3.3 Periocular dug derlivery route in AMD**

108 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

(ClinicalTrials.gov The MAP Study: Fluocinolone acetonide (FA)/MedidurTM for age related macular degeneration (AMD) Pilot. (2010). Available online: http://clinicaltrials.gov/ct2/show/ NCT00605423?term=Iluvien&rank=12), dry AMD (ClinicalTrials.gov Fluocinolone acetonide intravitreal inserts in geographic atrophy. (2010). Available online: http://clinicaltrials.gov/ct2/show/NCT00695318?term=Iluvien&rank=15. Brimonidine is an a2 adrenergic agonist, which can release various neurotrophins including brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) (Lonngren, Napankangas, Lafuente, et al. 2006; Kim, Chang, Kim, et al. 2007). These neurotrophins have a potential to prevent apoptosis of photoreceptors and/or RPE (Azadi, Johnson, Paquet-Durand, et al. 2007; Zhang, Mo, Fang, et al. 2009). PLGA intravitreal implant with two doses (200 mg, 400 mg) of brimonidine tartrate similar to Ozurdex® is now in Phase II clinical study for dry AMD (ClinicalTrials.gov Safety and efficacy of brimonidine intravitreal implant in patients with geographic atrophy due to age-related macular degeneration (AMD). (2010). Available online: http://clinicaltrials.gov/ct2/show/NCT00658619), and phase I/II clinical trials for RP (ClinicalTrials.gov An exploratory study to evaluate the safety of brimonidine intravitreal implant in patients with retinitis pigmentosa. (2010). Available online: http://clinicaltrials.gov/ct2/show/ NCT00661479) by Allergan, Inc. Neurotech Pharmaceuticals, Inc. (Lincoln, RI, U.S.) has been developing ―"Encapsulated Cell Technology", this is an implantable intravitreal device that uses genetically modified ARPE-19 RPE cells (transfected with plasmid gene insertion techniques) to release a therapeutic growth factor protein, ciliary neurotrophic factor (CNTF), with zero-order kinetics. CNTF is a neurotrophic growth factor with profound antiapoptotic effects that prolong the lives of dying photoreceptor cells. The cells are encapsulated within a semipermeable, hollow-fiber membrane, intravitreal implant device so that the CNTF protein is released to ocular tissues but the modified RPE cells are immunologically isolated from the patient, avoiding immune rejection. Phase 2 trials showed that this delivery system was safe, and a dose-dependent biological effect on the retina was observed. Potential visual benefit was also shown in patients with geographic atrophy (US National Institutes of Health. ClinicalTrials.org. NCT00447954 CgI. A Study of an Encapsulated Cell Technology (ECT) Implant for Patients With Atrophic Macular Degeneration. Phase II study. (2009). http://clinicaltrials.gov/ct2/show/NCT00447954). The duration of action of this encapsulated cell therapy device was found to be up to 2 years. Next-phase clinical trials are under way to continue to evaluate encapsulated cell technology (ECT)-CNTF's effectiveness

for the treatment of dry AMD and retinitis pigmentosa.

Almazan, et al. 2010).

Anti-VEGF compounds are now required to be administered into the eye every 4 to 6 weeks. Moreover, vitrectomized eyes highly increase the clearance rate of injected drugs and are, therefore, refractory to these drugs. The companies are currently interested in new methods of prolongation of residence time of intravitreally-injected drugs such as ranibizumab and pegaptanib. Viscous medium such as hyaluronic acid or gelling solution are considered as an additive to prolong drug residence in the vitreous cavity (Table 3) (Lyons, Ma & Trogde, 2009; Giese, Kaufmann & Klippel-Giese, 2004; McSwiggen, Beigelman, Macejak, et al. 2003; Robinson, Blanda, Hughes, et al. 2010; Robinson, Blanda, Liu, et al. 2010; Robinson, Tsai, Periocular delivery includes such avenues as subconjunctival, retrobulbar, peribulbar, sub-Tenon's and intrascleral delivery. This route requires drugs to pass through several layers of ocular tissue (episclera, sclera, choroid, Bruch's membrane, and RPE) to reach the retina or vitreous humor. Current knowledge shows that the combined effects of several static anatomic barriers and dynamic clearance mechanisms generally make periocular drug delivery one of the least effective ways of attaining high peak therapeutic intraocular drug concentrations in the retina or vitreous. These barriers are categorized into three major groups: static, dynamic and metabolic. Static barriers include the tissues that must be penetrated (e.g., sclera, Bruch's membrane-choroid and RPE). Dynamic barriers include blood flow, lymphatic drainage, transport proteins of the RPE, drug efflux pumps, organic ion transporters and bulk fluid flow from intraocular drainage systems. Metabolic barriers include enzyme systems such as cytochrome P450 and lysosomal enzymes, which have the ability to degrade or detoxify drugs. In addition to the static, dynamic and metabolic barriers, other factors must be considered in periocular delivery such the individual pharmacokinetic properties of the drug (molecular dimensions, molecular weight, atomic charge and chemical components of the drug). Taken together these factors result in low intraocular bioavailability of drug delivered by the various periocular drug techniques, compared with that of intravitreal injection.

Overall, when the safety and efficacy of this drug delivery route is compared with that of the others for the treatment of disease in the inner coat of the posterior segment, periocular injection is one of the best for safety, and it is in the middle to low end for efficacy, after intrinsic drug properties are factored in, such as potency and exposure–response relationships. With a few modifications, this route has the greatest potential to surpass intravitreal injection as the preferred treatment option for disease of the inner coat of the eye, since it deposits drug locally immediately adjacent to the targeted tissue without being overly invasive.

A major adjuvant used to overcome the short to medium duration of action of periocular drug solutions is the development of several sustained drug release systems, whether through formulation modifications or through various sustained release drug delivery devices. Many of these options include liposomes, microspheres, and microcapsules with

Basic Research and Clinical Application

**3.4 Hybrid drug delivery in AMD** 

technology.

**3.5 Topical drug delivery in AMD** 

mediated influx transporters.

has also been developed for suprachoroidal drug delivery.

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 111

Minimally invasive hollow and solid microneedles (<1 mm diameter) have been developed to deliver drugs into the cornea, sclera, or suprachoroidal space to avoid some of the shortcomings in safety and bioavailability with intravitreal or periocular injection. Solid, drug-coated microneedles are used for intracorneal and intrascleral drug delivery, improved bioavailability, and duration of action. Hollow microneedles are used for intrascleral and suprachoroidal delivery of a sustained-release drug depot in a tissue layer, with clearance mechanisms that are minimal or less than those in the subconjunctival or sub-Tenon's space. Microneedles allow for better retinochoroidal targeting than periocular drug delivery, because it is closer to the target tissue (Jiang, Gill, Ghate, et al. 2007; Jiang, Moore, Edelhauser, et al. 2009). A hollow microcatheter cannulation drug delivery technique

It is more directly invasive than the hollow microneedle approach, but microcatheter delivery may be promising for sustained drug delivery if it can be shown that continuous infusion of drugs into the suprachoroidal space can be tolerated better than a one-time injection with a microneedle. Overall, despite being relatively new and having few longterm data, the hybrid delivery route is arguably the second best for safety (tied with periocular and just behind topical) and second best for efficacy (the best being intraocular). This ranking has not been definitively proven in clinical trials. Another drawback that has not yet been calculated is the greater expense associated with this

Topical application to the anterior eye has been proven successful in the treatment of diseases owing to easy access to the target site. However, the adoption of mechanisms in ensuring topical drug penetration to the posterior eye presents numerous challenges. It is difficult to predict which drugs can achieve adequate therapeutic levels in the inner coat of the posterior segment after topical drug delivery and whether penetration can be enhanced by structural modifications or a particular formulation (Ghate & Edelhauser, 2006). Thus, experimental testing in animal models is critical. As this drug delivery route has two major shortcomings, extremely poor bioavailability to the inner coat of the posterior segment and a short duration of action, the main adjuvants are penetration enhancers and sustained-release drug delivery systems. Paracellular penetration by topical drugs can be improved by several mechanisms including: opening TJs by using preservatives in topical medications or by iatrogenic epithelial scraping; increasing drug lipophilicity through the use of prodrugs or other analogues, such as surfactants, and binding the drugs to dendrimers that use carrier-

Recent research has focused on small-molecule penetration into the vitreous, with evidence that molecules with lower molecular weight have increased permeability into the posterior chamber. Molecules with higher molecular weights and superior water solubility (highly charged) may have longer half-lives than those with lower molecular weights. Thus, lower molecular-weight compounds have increased access to the posterior eye and may minimize the risk of toxicity compared with higher molecular-weight compounds, which degrade at

diameters of 1–1000 μm, as well as nanospheres and nanocapsules with diameters of less than 1 μm, and biodegradable fibrin sealants (Bourges, Gautier, Delie, et al. 2003; Bu, Gukasyan, Goulet, et al. 2007; Guidetti, Azema, Malet-Martino, et al. 2008; Kearns & Williams, 2009). Polymeric microspheres have been used to target the RPE. Moritera et al. studied the use of surface-modified polymeric microspheres to localize drugs to the RPE (Moritera, Ogura, Yoshimura, et al. 1994). Phagocytosis by RPE was tracked by incorporating fluorescent dye into PLA microspheres with the rate of phagocytosis enhanced with gelatin-precoating as compared with bare microspheres. Intracellular dye release occurred following phagocytosis and could be controlled by varying the polymer formulation of the microspheres. Tuovinen et al. studied targeting drugs to the RPE using microparticles (11 μm in diameter) produced with starch acetate, which degrades more slowly than native starch (Tuovinen, Ruhanen, Kinnarinen, et al. 2004). These microparticles could be phagocytosed and degraded within the RPE.

Nanospheres have also been used to target the RPE for sustained drug delivery. Sakurai et al. studied the intraocular kinetics of nanospheres and found that polystyrene nanospheres containing fluorescein (2 μm in diameter) were detectable in the retina, vitreous and trabecular meshwork more than 1 month following an intravitreal injection in vivo in rabbits (Sakurai, Ozeki, Kunou, et al. 2001). Anionic nanoparticles traversed the collagen fibrils of the vitreous more readily than the cationic nanoparticles, showing potential as drug delivery vehicles for the subretinal space and the RPE. Muller cells take up the nanoparticles, possibly playing a key role in retinal penetration. Micro- and nanoparticles have potential in the field of gene therapy by functioning as nonviral vectors to enable cellular penetration, guard against degradation and maintain sustained delivery. Bejjani et al. explored the use of PLA and PLGA nanoparticles as vectors for gene transfer to a bovine and a human ARPE-19 cell line (Bejjani, BenEzra, Cohen, et al. 2005), concluding that PLGA could successfully sequester and internalize plasmids, resulting in gene expression in RPE detectable 48 h postinjection and maintained for 8 days. Another therapeutic approach is the inhibition of gene expression using antisense oligonucleotides, aptamers and siRNA (Fattal & Bochot, 2006; Fattal & Bochot, 2008; Tanito, Li, Elliott, et al. 2007). Aukunuru et al. showed that nanoparticles formulated using a PLGA (50:50) copolymer could deliver VEGF antisense oligonucleotide to the human ARPE-19 cell line, and inhibit VEGF secretion and mRNA expression (Aukunuru, Ayalasomayajula & Kompella, 2003). In a study performed by Carrasquillo et al. (Carrasquillo, Ricker, Rigas, et al. 2003) and summarized by Moshfeghi and Peyman (Moshfeghi & Peyman, 2005), the anti-VEGF RNA aptamer (EYE001, Macugen®, OSI Pharmaceuticals, NY, USA) was incorporated into PLGA microspheres to develop a sustainedrelease inhibition of VEGF for the treatment of neovascular AMD. Transport and drug efficacy studies of micro- and nanoparticles administered via periocular injection have been also published (Amrite, Edelhauser, Singh, et al. 2008; Amrite & Kompella, 2005; Ayalasomayajula & Kompella, 2003; Ayalasomayajula & Kompella, 2004; Ayalasomayajula & Kompella, 2005; Chiang, Tung & Lu, 2001; Saishin, Silva, Saishin, et al. 2003). In conclusion nano- and microparticles have shown great potential for expanding the arsenal of drug-delivery systems available for treating posterior segment disease. They provide sustained delivery and reduce complications that result from treatments requiring multiple injections. Transscleral delivery of anti-VEGF drugs loaded in PLA or PLGA nanoand microparticles is gaining much attention as a feasible and effective method of administration for the treatment of posterior segment disease.

#### **3.4 Hybrid drug delivery in AMD**

110 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

diameters of 1–1000 μm, as well as nanospheres and nanocapsules with diameters of less than 1 μm, and biodegradable fibrin sealants (Bourges, Gautier, Delie, et al. 2003; Bu, Gukasyan, Goulet, et al. 2007; Guidetti, Azema, Malet-Martino, et al. 2008; Kearns & Williams, 2009). Polymeric microspheres have been used to target the RPE. Moritera et al. studied the use of surface-modified polymeric microspheres to localize drugs to the RPE (Moritera, Ogura, Yoshimura, et al. 1994). Phagocytosis by RPE was tracked by incorporating fluorescent dye into PLA microspheres with the rate of phagocytosis enhanced with gelatin-precoating as compared with bare microspheres. Intracellular dye release occurred following phagocytosis and could be controlled by varying the polymer formulation of the microspheres. Tuovinen et al. studied targeting drugs to the RPE using microparticles (11 μm in diameter) produced with starch acetate, which degrades more slowly than native starch (Tuovinen, Ruhanen, Kinnarinen, et al. 2004). These microparticles

Nanospheres have also been used to target the RPE for sustained drug delivery. Sakurai et al. studied the intraocular kinetics of nanospheres and found that polystyrene nanospheres containing fluorescein (2 μm in diameter) were detectable in the retina, vitreous and trabecular meshwork more than 1 month following an intravitreal injection in vivo in rabbits (Sakurai, Ozeki, Kunou, et al. 2001). Anionic nanoparticles traversed the collagen fibrils of the vitreous more readily than the cationic nanoparticles, showing potential as drug delivery vehicles for the subretinal space and the RPE. Muller cells take up the nanoparticles, possibly playing a key role in retinal penetration. Micro- and nanoparticles have potential in the field of gene therapy by functioning as nonviral vectors to enable cellular penetration, guard against degradation and maintain sustained delivery. Bejjani et al. explored the use of PLA and PLGA nanoparticles as vectors for gene transfer to a bovine and a human ARPE-19 cell line (Bejjani, BenEzra, Cohen, et al. 2005), concluding that PLGA could successfully sequester and internalize plasmids, resulting in gene expression in RPE detectable 48 h postinjection and maintained for 8 days. Another therapeutic approach is the inhibition of gene expression using antisense oligonucleotides, aptamers and siRNA (Fattal & Bochot, 2006; Fattal & Bochot, 2008; Tanito, Li, Elliott, et al. 2007). Aukunuru et al. showed that nanoparticles formulated using a PLGA (50:50) copolymer could deliver VEGF antisense oligonucleotide to the human ARPE-19 cell line, and inhibit VEGF secretion and mRNA expression (Aukunuru, Ayalasomayajula & Kompella, 2003). In a study performed by Carrasquillo et al. (Carrasquillo, Ricker, Rigas, et al. 2003) and summarized by Moshfeghi and Peyman (Moshfeghi & Peyman, 2005), the anti-VEGF RNA aptamer (EYE001, Macugen®, OSI Pharmaceuticals, NY, USA) was incorporated into PLGA microspheres to develop a sustainedrelease inhibition of VEGF for the treatment of neovascular AMD. Transport and drug efficacy studies of micro- and nanoparticles administered via periocular injection have been also published (Amrite, Edelhauser, Singh, et al. 2008; Amrite & Kompella, 2005; Ayalasomayajula & Kompella, 2003; Ayalasomayajula & Kompella, 2004; Ayalasomayajula & Kompella, 2005; Chiang, Tung & Lu, 2001; Saishin, Silva, Saishin, et al. 2003). In conclusion nano- and microparticles have shown great potential for expanding the arsenal of drug-delivery systems available for treating posterior segment disease. They provide sustained delivery and reduce complications that result from treatments requiring multiple injections. Transscleral delivery of anti-VEGF drugs loaded in PLA or PLGA nanoand microparticles is gaining much attention as a feasible and effective method of

could be phagocytosed and degraded within the RPE.

administration for the treatment of posterior segment disease.

Minimally invasive hollow and solid microneedles (<1 mm diameter) have been developed to deliver drugs into the cornea, sclera, or suprachoroidal space to avoid some of the shortcomings in safety and bioavailability with intravitreal or periocular injection. Solid, drug-coated microneedles are used for intracorneal and intrascleral drug delivery, improved bioavailability, and duration of action. Hollow microneedles are used for intrascleral and suprachoroidal delivery of a sustained-release drug depot in a tissue layer, with clearance mechanisms that are minimal or less than those in the subconjunctival or sub-Tenon's space. Microneedles allow for better retinochoroidal targeting than periocular drug delivery, because it is closer to the target tissue (Jiang, Gill, Ghate, et al. 2007; Jiang, Moore, Edelhauser, et al. 2009). A hollow microcatheter cannulation drug delivery technique has also been developed for suprachoroidal drug delivery.

It is more directly invasive than the hollow microneedle approach, but microcatheter delivery may be promising for sustained drug delivery if it can be shown that continuous infusion of drugs into the suprachoroidal space can be tolerated better than a one-time injection with a microneedle. Overall, despite being relatively new and having few longterm data, the hybrid delivery route is arguably the second best for safety (tied with periocular and just behind topical) and second best for efficacy (the best being intraocular). This ranking has not been definitively proven in clinical trials. Another drawback that has not yet been calculated is the greater expense associated with this technology.

#### **3.5 Topical drug delivery in AMD**

Topical application to the anterior eye has been proven successful in the treatment of diseases owing to easy access to the target site. However, the adoption of mechanisms in ensuring topical drug penetration to the posterior eye presents numerous challenges. It is difficult to predict which drugs can achieve adequate therapeutic levels in the inner coat of the posterior segment after topical drug delivery and whether penetration can be enhanced by structural modifications or a particular formulation (Ghate & Edelhauser, 2006). Thus, experimental testing in animal models is critical. As this drug delivery route has two major shortcomings, extremely poor bioavailability to the inner coat of the posterior segment and a short duration of action, the main adjuvants are penetration enhancers and sustained-release drug delivery systems. Paracellular penetration by topical drugs can be improved by several mechanisms including: opening TJs by using preservatives in topical medications or by iatrogenic epithelial scraping; increasing drug lipophilicity through the use of prodrugs or other analogues, such as surfactants, and binding the drugs to dendrimers that use carriermediated influx transporters.

Recent research has focused on small-molecule penetration into the vitreous, with evidence that molecules with lower molecular weight have increased permeability into the posterior chamber. Molecules with higher molecular weights and superior water solubility (highly charged) may have longer half-lives than those with lower molecular weights. Thus, lower molecular-weight compounds have increased access to the posterior eye and may minimize the risk of toxicity compared with higher molecular-weight compounds, which degrade at

Basic Research and Clinical Application

neovascularization.

**4. Conclusion** 

**5. References** 

complement inhibitors to the alternative pathway.

new solutions to the challenge of ocular drug delivery.

of Drug Delivery Systems for the Treatment of Age-Related Macular Degeneration 113

intravenous fusion protein complement receptor 2 and factor H (CR2-fH), to recognize and inhibit complement- activation products (Holers, 2008; Rohrer, Long, Coughlin, et al. 2009; Thurman & Holers, 2006). Complement receptor 2 recognizes C3d, a tissue-bound activation product of complementmediated inflammation (e.g., drusen), whereas the fH component of the fusion molecule is the most potent inhibitor of the alternative complement pathway. Some experimental studies have shown how the oxidative stress sensitizes RPE cells to complement-mediated attack by decreasing regulatory cell surface membrane-bound

That oxidative stress also alters RPE cells in such a way that soluble fH in the serum is less functionally protective (Thurman, Renner, Kunchithapautham, et al. 2009). Complementmediated attack of the RPE then results in sublytic activation of the membrane attack complex resulting in vascular endothelial growth factor VEGF release and breakdown of the oBRB (Thurman, Renner, Kunchithapautham, et al. 2009). In humans, this cascade of events can result in either dry geographic atrophy or wet AMD. Systemic CR2-fH therapy protects the retina using experimental mouse models of retinal degeneration and choroidal

Basic research and open clinical trials have provided breakthrough therapies for treatment of diseases of the posterior segment of the eye, such as the use of anti-VEGF agents for the treatment of AMD. More effective drugs and drug-delivery systems are needed to decrease the frequency of drug administration. Multiple drugs and drug delivery systems may be required to safely and successfully treat some conditions. However, effective treatment of posterior segment ophthalmic diseases represents a formidable challenge for scientists and clinicians in the ophthalmic pharmaceutical field. The challenges include the anatomic and physiologic barriers that can impede pharmacologically active levels of drug from reaching the targeted tissues inside the eye, immune reactions and clearance mechanisms to certain drugs and drug delivery materials, and the often irreversible nature of vision loss. A better understanding must be developed of the nature and effect of dynamic physiologic processes of the eye, such as clearance mechanisms and metabolism of drugs in specific tissue layers. Each static anatomic barrier encountered for each specific drug delivery technique must be better studied. Drug–protein or drug–pigment binding must be better characterized with needing of more research in formulation modifications that alter physicochemical properties of both new and old drugs, facilitating delivery through known paracellular and transcellular pathways. Ophthalmic drug delivery via nanotechnology-based products, development of ophthalmic gene delivery must be further explored, given the extensive potential of this technology. However a new "era" is coming, in which application of technological advances in vision science is progressing at a rapid rate. Advances in nanotechnology, gene therapy, and biomaterials, for example, hold promise for providing

Acharya, N. & Young, L. (2004). Sustained-release drug implants for the treatment

ofintraocular disease. Int. Ophthalmol. Clin. 44:33–39.

slower rates. Therefore, each drug must be individually assessed and its uptake, efficiency and safety must be determined. As the list of drugs that achieve therapeutic levels in the retina and choroid after topical administration increases, it may be possible to identify structural characteristics that promote ocular penetration and to specifically design drugs and/or prodrugs accordingly. Another new technique is represented by Iontophoresis that is an old technology recently modified into a new innovative drug delivery platform. Recent clinical trials have demonstrated that iontophoresis is sufficiently safe and capable of delivering steroids to ocular tissue to some oculare disease (uveitis). It is performed by applying a small electrical current that has the same charge as the drug to create repulsive electromotive forces (Hayden, Jockovich, Murray, et al. 2006; Hughes, Olejnik, Chang-Lin, et al 2005; Myles, Neumann & Hill, 2005).

#### **3.6 Systemic drug delivery routes**

There have been advances in the use of systemic medications for the treatment of ophthalmic diseases, despite several limitations related to systemic penetration of many drugs, particularly large or hydrophilic ones, into the posterior segment of the eye. Further movement from the choroid into internal ocular structures such as the retina and vitreous humor, especially of large and/or hydrophilic drugs, is restricted by the RPE (oBRB) and TJs of the retinal vasculature (iBRB). Small lipophilic drugs, however, can penetrate the oBRB and iBRB, achieving appreciable concentrations in the retina and vitreous humor after systemic administration. The systemic application of drugs not only increases the quantity of a drug necessary to achieve therapeutic concentrations, but it also increases the risk of adverse effects due to the accumulation of a drug in other tissues throughout the body. Another limitation of systemic application includes potential reduced time of therapeutic effects and potency due to the dilution and degradation of the drug before reaching the target site. Despite these limitations, there have been advances in the use of systemic medications for the treatment of ophthalmic diseases.

To the wide sense, PDT with verteporfin is one of drug targeting systems. After intravenous administration, verteporfin, a relatively hydrophobic compound, is incorporated into lipoproteins. According to preferential accumulation of lipoproteins in and around choroidal neovascular membrane, verteporfin tends to be targeted into CNV. The photosensitizer targeted injures and closes new vessels in combination of laser irradiation to pathological lesion. However, most systemic drugs are now formulated with excipients that help overcome their tendency toward a brief duration of action and poor to medium bioavailability to the inner coat of the posterior segment.

One novel scheme to enhance the bioavailability of systemic drugs to the retina are penetration enhancers that help reversibly open up the BRB or improve transcellular penetration. Because TJ proteins are not very antigenic, it is difficult to develop antibodies against their extracellular domain, a fact that has severely hampered the development of TJ modulators. For example a novel approach to treat retinal disease by systemic drug delivery reviewed the role of the complement pathway in the pathogenesis of ARMD. Dysregulation of the alternate complement pathway, especially in the C3 amplification loop, may be a reasonable target for treating AMD and inflammatory retinal diseases by administering the intravenous fusion protein complement receptor 2 and factor H (CR2-fH), to recognize and inhibit complement- activation products (Holers, 2008; Rohrer, Long, Coughlin, et al. 2009; Thurman & Holers, 2006). Complement receptor 2 recognizes C3d, a tissue-bound activation product of complementmediated inflammation (e.g., drusen), whereas the fH component of the fusion molecule is the most potent inhibitor of the alternative complement pathway. Some experimental studies have shown how the oxidative stress sensitizes RPE cells to complement-mediated attack by decreasing regulatory cell surface membrane-bound complement inhibitors to the alternative pathway.

That oxidative stress also alters RPE cells in such a way that soluble fH in the serum is less functionally protective (Thurman, Renner, Kunchithapautham, et al. 2009). Complementmediated attack of the RPE then results in sublytic activation of the membrane attack complex resulting in vascular endothelial growth factor VEGF release and breakdown of the oBRB (Thurman, Renner, Kunchithapautham, et al. 2009). In humans, this cascade of events can result in either dry geographic atrophy or wet AMD. Systemic CR2-fH therapy protects the retina using experimental mouse models of retinal degeneration and choroidal neovascularization.

#### **4. Conclusion**

112 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

slower rates. Therefore, each drug must be individually assessed and its uptake, efficiency and safety must be determined. As the list of drugs that achieve therapeutic levels in the retina and choroid after topical administration increases, it may be possible to identify structural characteristics that promote ocular penetration and to specifically design drugs and/or prodrugs accordingly. Another new technique is represented by Iontophoresis that is an old technology recently modified into a new innovative drug delivery platform. Recent clinical trials have demonstrated that iontophoresis is sufficiently safe and capable of delivering steroids to ocular tissue to some oculare disease (uveitis). It is performed by applying a small electrical current that has the same charge as the drug to create repulsive electromotive forces (Hayden, Jockovich, Murray, et al. 2006; Hughes, Olejnik, Chang-Lin, et

There have been advances in the use of systemic medications for the treatment of ophthalmic diseases, despite several limitations related to systemic penetration of many drugs, particularly large or hydrophilic ones, into the posterior segment of the eye. Further movement from the choroid into internal ocular structures such as the retina and vitreous humor, especially of large and/or hydrophilic drugs, is restricted by the RPE (oBRB) and TJs of the retinal vasculature (iBRB). Small lipophilic drugs, however, can penetrate the oBRB and iBRB, achieving appreciable concentrations in the retina and vitreous humor after systemic administration. The systemic application of drugs not only increases the quantity of a drug necessary to achieve therapeutic concentrations, but it also increases the risk of adverse effects due to the accumulation of a drug in other tissues throughout the body. Another limitation of systemic application includes potential reduced time of therapeutic effects and potency due to the dilution and degradation of the drug before reaching the target site. Despite these limitations, there have been advances in the use of systemic

To the wide sense, PDT with verteporfin is one of drug targeting systems. After intravenous administration, verteporfin, a relatively hydrophobic compound, is incorporated into lipoproteins. According to preferential accumulation of lipoproteins in and around choroidal neovascular membrane, verteporfin tends to be targeted into CNV. The photosensitizer targeted injures and closes new vessels in combination of laser irradiation to pathological lesion. However, most systemic drugs are now formulated with excipients that help overcome their tendency toward a brief duration of action and poor to medium

One novel scheme to enhance the bioavailability of systemic drugs to the retina are penetration enhancers that help reversibly open up the BRB or improve transcellular penetration. Because TJ proteins are not very antigenic, it is difficult to develop antibodies against their extracellular domain, a fact that has severely hampered the development of TJ modulators. For example a novel approach to treat retinal disease by systemic drug delivery reviewed the role of the complement pathway in the pathogenesis of ARMD. Dysregulation of the alternate complement pathway, especially in the C3 amplification loop, may be a reasonable target for treating AMD and inflammatory retinal diseases by administering the

al 2005; Myles, Neumann & Hill, 2005).

medications for the treatment of ophthalmic diseases.

bioavailability to the inner coat of the posterior segment.

**3.6 Systemic drug delivery routes** 

Basic research and open clinical trials have provided breakthrough therapies for treatment of diseases of the posterior segment of the eye, such as the use of anti-VEGF agents for the treatment of AMD. More effective drugs and drug-delivery systems are needed to decrease the frequency of drug administration. Multiple drugs and drug delivery systems may be required to safely and successfully treat some conditions. However, effective treatment of posterior segment ophthalmic diseases represents a formidable challenge for scientists and clinicians in the ophthalmic pharmaceutical field. The challenges include the anatomic and physiologic barriers that can impede pharmacologically active levels of drug from reaching the targeted tissues inside the eye, immune reactions and clearance mechanisms to certain drugs and drug delivery materials, and the often irreversible nature of vision loss. A better understanding must be developed of the nature and effect of dynamic physiologic processes of the eye, such as clearance mechanisms and metabolism of drugs in specific tissue layers. Each static anatomic barrier encountered for each specific drug delivery technique must be better studied. Drug–protein or drug–pigment binding must be better characterized with needing of more research in formulation modifications that alter physicochemical properties of both new and old drugs, facilitating delivery through known paracellular and transcellular pathways. Ophthalmic drug delivery via nanotechnology-based products, development of ophthalmic gene delivery must be further explored, given the extensive potential of this technology. However a new "era" is coming, in which application of technological advances in vision science is progressing at a rapid rate. Advances in nanotechnology, gene therapy, and biomaterials, for example, hold promise for providing new solutions to the challenge of ocular drug delivery.

#### **5. References**

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**Part 2** 

**Clinical Research** 


## **Part 2**

**Clinical Research** 

120 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

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**7** 

*Croatia* 

**Treatment of Neovascular** 

Ratimir Lazić and Nikica Gabrić *University Eye Clinic Svjetlost Zagreb* 

**Age Related Macular Degeneration** 

Neovascular Age Related Macular Degeneration (nAMD) has been a therapeutic challenge until recently. The natural course of the disease leads to a great deterioration of visual acuity and is considered to be the leading cause of legal blindness in people 50 years of age or

Until the end of the last century, no intervention could alter the natural history of the disease. Only in the last few decades, the retina specialists began to intervene in order to minimize the visual loss in those patients. Last ten years have been especially exciting as the new treatment modalities emerged and for the first time we could not only halt the

In this chapter we will present therapeutic modalities which were applied chronologically and then we will present the up to date treatment options. Finally we will briefly summarize

The first major clinical trial evaluating laser photocoagulation in treatment of nAMD was performed in the 1980-ies, at times when no treatment modality could change natural course of this disease. However, the Macular Photocoagulation Study (MPS) (1), which took 5 years to complete, demonstrated that argon laser photocoagulation could postpone or even prevent significant visual loss in patients with juxtafoveol and extrafoveol choroidal

It was later shown that some benefits of reducing the damage generated by the natural course of the disease could be achieved by performing laser treatment on subfoveol CNV as well (2). As the reduction of visual acuity occurred immediately after the laser treatment,

Photodynamic therapy (PDT) represented the first specific treatment option in treating nAMD and the treatment protocol consisted of intravenous application of a photosensitive

progression of the deterioration, but rather improve vision in some patients.

new emerging drugs which are still under clinical evaluation.

**1. Introduction** 

**2. Past treatments** 

**2.1 Laser photocoagulation** 

neovascular membranes (CNV).

**2.2 Photodynamic therapy** 

patients' dissatisfaction became a major issue.

older, especially in the Western countries.

## **Treatment of Neovascular Age Related Macular Degeneration**

Ratimir Lazić and Nikica Gabrić *University Eye Clinic Svjetlost Zagreb Croatia* 

### **1. Introduction**

Neovascular Age Related Macular Degeneration (nAMD) has been a therapeutic challenge until recently. The natural course of the disease leads to a great deterioration of visual acuity and is considered to be the leading cause of legal blindness in people 50 years of age or older, especially in the Western countries.

Until the end of the last century, no intervention could alter the natural history of the disease. Only in the last few decades, the retina specialists began to intervene in order to minimize the visual loss in those patients. Last ten years have been especially exciting as the new treatment modalities emerged and for the first time we could not only halt the progression of the deterioration, but rather improve vision in some patients.

In this chapter we will present therapeutic modalities which were applied chronologically and then we will present the up to date treatment options. Finally we will briefly summarize new emerging drugs which are still under clinical evaluation.

#### **2. Past treatments**

#### **2.1 Laser photocoagulation**

The first major clinical trial evaluating laser photocoagulation in treatment of nAMD was performed in the 1980-ies, at times when no treatment modality could change natural course of this disease. However, the Macular Photocoagulation Study (MPS) (1), which took 5 years to complete, demonstrated that argon laser photocoagulation could postpone or even prevent significant visual loss in patients with juxtafoveol and extrafoveol choroidal neovascular membranes (CNV).

It was later shown that some benefits of reducing the damage generated by the natural course of the disease could be achieved by performing laser treatment on subfoveol CNV as well (2). As the reduction of visual acuity occurred immediately after the laser treatment, patients' dissatisfaction became a major issue.

#### **2.2 Photodynamic therapy**

Photodynamic therapy (PDT) represented the first specific treatment option in treating nAMD and the treatment protocol consisted of intravenous application of a photosensitive

Treatment of Neovascular Age Related Macular Degeneration 125

The VIP study (Verteporfin in Photodynamic Therapy) evaluated PDT in patients with occult CNV and showed that 121 (54%) patients from the verteporfin group lost fewer than 15 letters during 24 months of the follow-up, compared to 76 (67%) patients from the control group (8), whereby the best results were displayed by the patients with lesions smaller than

The mentioned studies demonstrated the efficacy of the PDT with verteporfin in maintaining the visual acuity or slowing down visual loss. It was the very first time that we

Surgical techniques in treatment of nAMD included submacular surgery, macular

Macular translocation was a surgical procedure involving the detachment of the retina along with macula in order to move or translocate fovea from the diseased RPE onto healthy RPE. Although there had been reports of case series with quite good visual outcomes after the surgery, severe complications could arise during the process of retinal displacement (10,11). Therefore nowadays macular translocation may not be considered for most patients with nAMD given the treatment options already available. In the anti-VEGF era macular translocation may be employed in patients either with very advanced AMD or in those

Unfortunately the surgery undertaken in a very advanced cases wouldn't result in significant improvement of vision as the degenerative process had already damaged the retinal macular tissue along with the diseased underlying RPE: but retina specialist are reluctant to recommend macular translocation at the stage of the disease while macular retina is still viable at which point macular translocation would result in better functional

The full macular translocation surgery involved a detachment of the entire retina from the RPE by subretinal infusion of balanced saline solution fluid via 40 gauge needle with 360 degrees circumferential retinotomy followed by the retinal rotation under perflorocarbon liquid during which process macula was displaced onto healthy RPE. The site of the 360 retinotomy and the holes artificially created for detaching retina were sealed by endolaser photocoagulation. Then an exchange between perflorocarbon liquid with silicone oil was

An additional step forward was made by introducing the anti-VEGF drugs as a treatment option for nAMD. Pegaptanib sodium (Macugen) was actually a synthetically derived polyonucleotide ligand binding specifically to VEGF-A 165 isoform. The affinity to only one isoform of VEGF cluster explained pegaptanib sodium inferior therapeutic effect, compared

4 disc diameters or with the initial visual acuity better than 20/50 (9).

translocation, and submacular hemorrhage displacement.

patients with disease recalcitrant to anti-VEGF therapy.

performed. After 3 months silicone oil was removed.

to non-selective infibitors of all VEGF isoforms.

**3. Current treatment options** 

**3.1 Anti-VEGF drugs** 

**3.1.1 Pegaptanib sodium** 

**2.3 Surgical treatment** 

outcome.

could positively interfere with the natural course of the disease using PDT.

drug verteporfin, which was then activated by a 693-nanometer-long, verteporfin-sensitive laser beam light.

Verterporfin activation was followed by a series of photochemical reactions, resulting in destruction and thrombosis of endothelial cells in the neovascular membrane complex (3). The PDT had a double selectivity mechanism:


With verteporfin accumulating itself selectively within the neovascular complex, the collateral damage to surrounding healthy tissue was minimal. Verteporfin was in circulation carried by plasma lipoproteins, which were utilized by endothelial cells due to accelerated metabolism of neovascular complex. Within the area illuminated by the laser beam, shortacting free oxygen radicals induced endothelial cell damage, which process occurred through lipo and cyclooxigenase modulated pathways, the final result being trombocyte aggregation and vasoconstriction of neovascular complex vessels (4). The slow metabolism of normal blood vessels made the utilization of verteporfin-carrying lipoproteins in normal vessels slow as well. Verterporfin was utilized by neovascular complex vessels within 15 minutes. It took 30 minutes for normal blood vessels to utilize verteporfin. Therefore, the laser beam of 90 seconds duration must had been applied to the neovascular membrane no later than 30 minutes after the infusion was started, in order to prevent damage to normal blood vessels. The exact size and location of neovascular complex was determined by fluorescein angiography, which was a precondition to laser application.

Treatment of AMD with Photodynamic Therapy (TAP) study (5) was a double blind placebo controlled randomized clinical trial evaluating the efficacy of PDT in treatment of minimally classic CNV. 609 patients were enrolled (402 in the verteporfin group and 207 in the placebo group). The treatment was repeated every 3 months in case of relapse of leakage or continuous leakage, which was confirmed by fluorescein angiography. At 12 month followup 61.2% of patients from the verteporfin group, compared to 46.4% patients from the placebo group lost less than 3 logMar lines (minimal angle resolution logarithm) or 15 letters which was statistically significant. At 24 months follow-up, 213 (53%) patients from the verteporfin group, compared to 78 (38%) patients from the placebo group lost fewer than 3 lines (6). The mean visual acuity was 1.3 lines better in the verteporfin group. The eyes treated with verteporfin had a 16% better chance for visual improvement by 1 or more lines, compared to the placebo group. The benefits of the verteporfin treatment were higher in case of the predominantly CNV (at least 50% of the neovascular complex has a classic component): 33% of patients from the verteporfin group lost 3 or more lines, compared to 61% of patients from the placebo group after 12 months. If CNV was completely classic, the results were even better: 23% of patients from the verteporfin group lost fewer than 3 lines, whereas in the placebo group, 73% of patients lost 3 or more lines.

In case of the minimally classic type of the CNV, results were however modest **-** positive effects of the treatment were noticed only if the maximal diameter of neovascular lesion was smaller than 4 disc diameters. The efficacy of the PDT for the minimally classic CNV with the greatest diameter of lesion up to 6 discs was later confirmed by the VIM study (Verteporfin Therapy of Subfoveal Minimally Classic CNV in AMD) (7).

The VIP study (Verteporfin in Photodynamic Therapy) evaluated PDT in patients with occult CNV and showed that 121 (54%) patients from the verteporfin group lost fewer than 15 letters during 24 months of the follow-up, compared to 76 (67%) patients from the control group (8), whereby the best results were displayed by the patients with lesions smaller than 4 disc diameters or with the initial visual acuity better than 20/50 (9).

The mentioned studies demonstrated the efficacy of the PDT with verteporfin in maintaining the visual acuity or slowing down visual loss. It was the very first time that we could positively interfere with the natural course of the disease using PDT.

#### **2.3 Surgical treatment**

124 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

drug verteporfin, which was then activated by a 693-nanometer-long, verteporfin-sensitive

Verterporfin activation was followed by a series of photochemical reactions, resulting in destruction and thrombosis of endothelial cells in the neovascular membrane complex (3).

1. Verteporfin was utilized only by cells with accelerated metabolism (neovascular

With verteporfin accumulating itself selectively within the neovascular complex, the collateral damage to surrounding healthy tissue was minimal. Verteporfin was in circulation carried by plasma lipoproteins, which were utilized by endothelial cells due to accelerated metabolism of neovascular complex. Within the area illuminated by the laser beam, shortacting free oxygen radicals induced endothelial cell damage, which process occurred through lipo and cyclooxigenase modulated pathways, the final result being trombocyte aggregation and vasoconstriction of neovascular complex vessels (4). The slow metabolism of normal blood vessels made the utilization of verteporfin-carrying lipoproteins in normal vessels slow as well. Verterporfin was utilized by neovascular complex vessels within 15 minutes. It took 30 minutes for normal blood vessels to utilize verteporfin. Therefore, the laser beam of 90 seconds duration must had been applied to the neovascular membrane no later than 30 minutes after the infusion was started, in order to prevent damage to normal blood vessels. The exact size and location of neovascular complex was determined by

Treatment of AMD with Photodynamic Therapy (TAP) study (5) was a double blind placebo controlled randomized clinical trial evaluating the efficacy of PDT in treatment of minimally classic CNV. 609 patients were enrolled (402 in the verteporfin group and 207 in the placebo group). The treatment was repeated every 3 months in case of relapse of leakage or continuous leakage, which was confirmed by fluorescein angiography. At 12 month followup 61.2% of patients from the verteporfin group, compared to 46.4% patients from the placebo group lost less than 3 logMar lines (minimal angle resolution logarithm) or 15 letters which was statistically significant. At 24 months follow-up, 213 (53%) patients from the verteporfin group, compared to 78 (38%) patients from the placebo group lost fewer than 3 lines (6). The mean visual acuity was 1.3 lines better in the verteporfin group. The eyes treated with verteporfin had a 16% better chance for visual improvement by 1 or more lines, compared to the placebo group. The benefits of the verteporfin treatment were higher in case of the predominantly CNV (at least 50% of the neovascular complex has a classic component): 33% of patients from the verteporfin group lost 3 or more lines, compared to 61% of patients from the placebo group after 12 months. If CNV was completely classic, the results were even better: 23% of patients from the verteporfin group lost fewer than 3 lines,

In case of the minimally classic type of the CNV, results were however modest **-** positive effects of the treatment were noticed only if the maximal diameter of neovascular lesion was smaller than 4 disc diameters. The efficacy of the PDT for the minimally classic CNV with the greatest diameter of lesion up to 6 discs was later confirmed by the VIM study

laser beam light.

membrane),

The PDT had a double selectivity mechanism:

2. Laser beam was applied only to the area of CNV.

fluorescein angiography, which was a precondition to laser application.

whereas in the placebo group, 73% of patients lost 3 or more lines.

(Verteporfin Therapy of Subfoveal Minimally Classic CNV in AMD) (7).

Surgical techniques in treatment of nAMD included submacular surgery, macular translocation, and submacular hemorrhage displacement.

Macular translocation was a surgical procedure involving the detachment of the retina along with macula in order to move or translocate fovea from the diseased RPE onto healthy RPE. Although there had been reports of case series with quite good visual outcomes after the surgery, severe complications could arise during the process of retinal displacement (10,11). Therefore nowadays macular translocation may not be considered for most patients with nAMD given the treatment options already available. In the anti-VEGF era macular translocation may be employed in patients either with very advanced AMD or in those patients with disease recalcitrant to anti-VEGF therapy.

Unfortunately the surgery undertaken in a very advanced cases wouldn't result in significant improvement of vision as the degenerative process had already damaged the retinal macular tissue along with the diseased underlying RPE: but retina specialist are reluctant to recommend macular translocation at the stage of the disease while macular retina is still viable at which point macular translocation would result in better functional outcome.

The full macular translocation surgery involved a detachment of the entire retina from the RPE by subretinal infusion of balanced saline solution fluid via 40 gauge needle with 360 degrees circumferential retinotomy followed by the retinal rotation under perflorocarbon liquid during which process macula was displaced onto healthy RPE. The site of the 360 retinotomy and the holes artificially created for detaching retina were sealed by endolaser photocoagulation. Then an exchange between perflorocarbon liquid with silicone oil was performed. After 3 months silicone oil was removed.

#### **3. Current treatment options**

#### **3.1 Anti-VEGF drugs**

#### **3.1.1 Pegaptanib sodium**

An additional step forward was made by introducing the anti-VEGF drugs as a treatment option for nAMD. Pegaptanib sodium (Macugen) was actually a synthetically derived polyonucleotide ligand binding specifically to VEGF-A 165 isoform. The affinity to only one isoform of VEGF cluster explained pegaptanib sodium inferior therapeutic effect, compared to non-selective infibitors of all VEGF isoforms.

Treatment of Neovascular Age Related Macular Degeneration 127

In PRONTO study (17) patients received 3 injections of ranibizumab on a monthly basis and thereafter as needed, based on strictly defined criteria, which included visual acuity decline, reoccurrence of fluid and central retinal thickness increase measured by OCT and clinical manifestation of macular hemorrhage. The results obtained by the PRONTO study were comparable to those of the MARINA and ANCHOR studies, but the number of injections required was lower: 5 injection of ranibizumab in the PRONTO versus 12 injections in the

SAILOR study (18) was designed with the purpose of confirming the results obtained by the PRONTO study. The SAILOR study lasted for 12 months. After three monthly injections of ranibizumab the additional injections were given based on predefined criteria, similar to those applied in the PRONTO study. The results of the SAILOR study were better than the ones of the PIER study, but not as good as those of the MARINA and ANCHOR, where injections were given on a monthly basis. The rate of the systemic side effects indicated a

SUSTAIN study (19) confirmed the results given by the SAILOR study. The SUSTAIN study achieved better results than the PIER study, but inferior to those of the MARINA and ANCHOR and the PRONTO studies. During a 1-year follow-up period, mean number of injections applied was around 5 and visual acuity improved by 3.6 letters. Central retinal

The HORIZON study (20) was a sequel to the MARINA and ANCHOR studies, its main purpose was to analyze the long term follow-up results and furthermore, to switch from monthly dosing to dosing as needed and to observe an impact on visual acuity. Additional treatment was required in more than 60% of patients during the 2 year follow-up period. Visual gain achieved after two years of monthly injections of ranibizumab was not

The monthly dosing remained, however, the best way to preserve the visual acuity. Any reduction in number of injections led to inferior visual outcome. Due to frequent dosing and other paramedical factors, regular monthly drug application presented a great burden to

Bevacizumab (Avastin) is a humanized recombinant monoclonal mouse antibody**.** A molecular weight of bevacizumab is 149 kilodaltons. It is active against all VEGF-A isoforms. Bevacizumab has been approved as an intravenous drug in the treatment of metastatic colon cancer (21), along with chemotherapy. It helps to induce the reduction of

In 2005, bevacizumab was administered as ocular treatment for the first time. First bevacizumab trial included 9 patients with nAMD. Bevacizumab was given intravenously (5mg/kg) at two week intervals resulting in 12-letters gain with a significant reduction of intraretinal macular edema at the 12 week follow-up period (23). 7 patients experienced

Due to systemic side-effects (hypertension, gastrointestinal bleeding, thromboembolic events), bevacizumab was then applied intravitrealy in a concentration of 1 mg/0.1 ml, which was a significantly lower dose compared to the systemic one, while the good visual

MARINA and the ANCHOR per year.

thickness was reduced by 92 microns.

substantial number of patients.

mild hypertension as a side-effect.

**3.1.3 Bevacizumab** 

maintained with less frequent dosing of ranibizumab.

the tumor volume by deprivation of tumor vascularization (22).

satisfactory safety profile.

Data from VISION trial indicated that 70% of the treated patients with intravitreal pegaptanib sodium given on a 6-week basis lost fewer than 15 letters during a 24 month follow-up, compared to 55% of patients from the placebo group. The mean visual acuity decreased by 8 letters during the follow-up period in the patients treated with intravitreal pegaptanib sodium in this study (12).

#### **3.1.2 Ranibizumab**

Ranibizumab is a humanized antigen-binding FAB fragment monoclonal antibody towards human VEGF-A, derived from rodents. It is produced by recombinant technology from Escherichiae coli, with molecular weight of 48 kilodaltons. Ranibizumab inhibits all VEGF-A isoforms. Furthermore, it penetrates well through all layers of retina up to choroidea. The binding affinity of ranibizumab is 5-20 times higher comparing to bevacizumab(13). Ranibizumab was approved for intravitreal application in 2006.

A large multicentric randomized clinical trial MARINA (Ranibizumab in Treatment of Occult and Minimally Classic CNV)(14) and ANCHOR (Ranibizumab in Treatment of Predominantly Classic CNV)(15) confirmed that ranibizumab, given on a monthly basis substantially improved visual acuity compared to placebo. The MARINA study enrolled 716 patients and evaluated the efficacy of monthly ranibizumab (0.3mg and 0.5 mg), versus placebo given for 24 months. In the ranibizumab groups over 90% of patients lost fewer than 15 letters (or 3 lines) compared to 62% patients from the placebo group. One third of patients from the ranibizumab group (0.5mg) gained 15 letters or more, while in the placebo group only 5% of patients achieved this effect. This was a significant breakthrough compared to all previous treatment options. Mean visual acuity improved by 7.2 letters (about 1 line of logMAR) at 12 month follow-up in the ranibizumab group (0.5mg), while in the placebo group, the visual acuity deteriorated by more than 10 letters. The benefit in visual acuity was maintained at 24 months. During 24 months, presumed endophthalmitis was identified in five patients (1.0%) and serious uveitis in six patients (1.3%).

The ANCHOR study enrolled 423 patients and compared the efficacy and safety of ranibizumab given on a monthly basis with standard PDT with verteporfin. The follow-up period was 24 months. At the end of the study, over 90% of patients from the ranibizumab group and 65% of patients from the photodynamic group lost fewer than 15 letters. Furthermore, over one third of patients from the ranibizumab group gained 15 letters or more, compared to 5.6% of patients from the PDT group. Mean visual acuity improved by 11.3 letters in the ranibizumab group (0.5mg), whereas patients from the photodynamic group had mean decrease of 9.5 letters. Reduction in central retinal thickness, measured by OCT, was also observed through the follow-up.

Endophthalmitis or serious uveitis occurred in around 2% of patients from the ranibizumab group (0.5 mg). Even though visual results were good, it took 24 injections over 2 year period to achieve it. Several studies were conducted in order to see whether a number of retreatments could be reduced with sustained visual results.

PIER study (16) analyzed a treatment protocol of three consecutive monthly injections of ranibizumab, followed by injections every three months. The results of this study were inferior when compared to the results achieved by the MARINA and ANCHOR trials, in particular between 3rd and 12th month when injections were given quarterly.

Data from VISION trial indicated that 70% of the treated patients with intravitreal pegaptanib sodium given on a 6-week basis lost fewer than 15 letters during a 24 month follow-up, compared to 55% of patients from the placebo group. The mean visual acuity decreased by 8 letters during the follow-up period in the patients treated with intravitreal

Ranibizumab is a humanized antigen-binding FAB fragment monoclonal antibody towards human VEGF-A, derived from rodents. It is produced by recombinant technology from Escherichiae coli, with molecular weight of 48 kilodaltons. Ranibizumab inhibits all VEGF-A isoforms. Furthermore, it penetrates well through all layers of retina up to choroidea. The binding affinity of ranibizumab is 5-20 times higher comparing to bevacizumab(13).

A large multicentric randomized clinical trial MARINA (Ranibizumab in Treatment of Occult and Minimally Classic CNV)(14) and ANCHOR (Ranibizumab in Treatment of Predominantly Classic CNV)(15) confirmed that ranibizumab, given on a monthly basis substantially improved visual acuity compared to placebo. The MARINA study enrolled 716 patients and evaluated the efficacy of monthly ranibizumab (0.3mg and 0.5 mg), versus placebo given for 24 months. In the ranibizumab groups over 90% of patients lost fewer than 15 letters (or 3 lines) compared to 62% patients from the placebo group. One third of patients from the ranibizumab group (0.5mg) gained 15 letters or more, while in the placebo group only 5% of patients achieved this effect. This was a significant breakthrough compared to all previous treatment options. Mean visual acuity improved by 7.2 letters (about 1 line of logMAR) at 12 month follow-up in the ranibizumab group (0.5mg), while in the placebo group, the visual acuity deteriorated by more than 10 letters. The benefit in visual acuity was maintained at 24 months. During 24 months, presumed endophthalmitis

was identified in five patients (1.0%) and serious uveitis in six patients (1.3%).

OCT, was also observed through the follow-up.

retreatments could be reduced with sustained visual results.

The ANCHOR study enrolled 423 patients and compared the efficacy and safety of ranibizumab given on a monthly basis with standard PDT with verteporfin. The follow-up period was 24 months. At the end of the study, over 90% of patients from the ranibizumab group and 65% of patients from the photodynamic group lost fewer than 15 letters. Furthermore, over one third of patients from the ranibizumab group gained 15 letters or more, compared to 5.6% of patients from the PDT group. Mean visual acuity improved by 11.3 letters in the ranibizumab group (0.5mg), whereas patients from the photodynamic group had mean decrease of 9.5 letters. Reduction in central retinal thickness, measured by

Endophthalmitis or serious uveitis occurred in around 2% of patients from the ranibizumab group (0.5 mg). Even though visual results were good, it took 24 injections over 2 year period to achieve it. Several studies were conducted in order to see whether a number of

PIER study (16) analyzed a treatment protocol of three consecutive monthly injections of ranibizumab, followed by injections every three months. The results of this study were inferior when compared to the results achieved by the MARINA and ANCHOR trials, in

particular between 3rd and 12th month when injections were given quarterly.

Ranibizumab was approved for intravitreal application in 2006.

pegaptanib sodium in this study (12).

**3.1.2 Ranibizumab** 

In PRONTO study (17) patients received 3 injections of ranibizumab on a monthly basis and thereafter as needed, based on strictly defined criteria, which included visual acuity decline, reoccurrence of fluid and central retinal thickness increase measured by OCT and clinical manifestation of macular hemorrhage. The results obtained by the PRONTO study were comparable to those of the MARINA and ANCHOR studies, but the number of injections required was lower: 5 injection of ranibizumab in the PRONTO versus 12 injections in the MARINA and the ANCHOR per year.

SAILOR study (18) was designed with the purpose of confirming the results obtained by the PRONTO study. The SAILOR study lasted for 12 months. After three monthly injections of ranibizumab the additional injections were given based on predefined criteria, similar to those applied in the PRONTO study. The results of the SAILOR study were better than the ones of the PIER study, but not as good as those of the MARINA and ANCHOR, where injections were given on a monthly basis. The rate of the systemic side effects indicated a satisfactory safety profile.

SUSTAIN study (19) confirmed the results given by the SAILOR study. The SUSTAIN study achieved better results than the PIER study, but inferior to those of the MARINA and ANCHOR and the PRONTO studies. During a 1-year follow-up period, mean number of injections applied was around 5 and visual acuity improved by 3.6 letters. Central retinal thickness was reduced by 92 microns.

The HORIZON study (20) was a sequel to the MARINA and ANCHOR studies, its main purpose was to analyze the long term follow-up results and furthermore, to switch from monthly dosing to dosing as needed and to observe an impact on visual acuity. Additional treatment was required in more than 60% of patients during the 2 year follow-up period. Visual gain achieved after two years of monthly injections of ranibizumab was not maintained with less frequent dosing of ranibizumab.

The monthly dosing remained, however, the best way to preserve the visual acuity. Any reduction in number of injections led to inferior visual outcome. Due to frequent dosing and other paramedical factors, regular monthly drug application presented a great burden to substantial number of patients.

#### **3.1.3 Bevacizumab**

Bevacizumab (Avastin) is a humanized recombinant monoclonal mouse antibody**.** A molecular weight of bevacizumab is 149 kilodaltons. It is active against all VEGF-A isoforms. Bevacizumab has been approved as an intravenous drug in the treatment of metastatic colon cancer (21), along with chemotherapy. It helps to induce the reduction of the tumor volume by deprivation of tumor vascularization (22).

In 2005, bevacizumab was administered as ocular treatment for the first time. First bevacizumab trial included 9 patients with nAMD. Bevacizumab was given intravenously (5mg/kg) at two week intervals resulting in 12-letters gain with a significant reduction of intraretinal macular edema at the 12 week follow-up period (23). 7 patients experienced mild hypertension as a side-effect.

Due to systemic side-effects (hypertension, gastrointestinal bleeding, thromboembolic events), bevacizumab was then applied intravitrealy in a concentration of 1 mg/0.1 ml, which was a significantly lower dose compared to the systemic one, while the good visual

Treatment of Neovascular Age Related Macular Degeneration 129

and VEGFR2 similar domains combined with Fc immunoglobulin segment (36). This means that VEGF Trap Eye has antibody inhibiting characteristics for all VEGF-A isoforms and binding affinity 800 times greater than bevacizumab. A clinical trial with 25 patients has shown good tolerability and increased visual acuity 6 weeks after single intravitreal

VIEW 1 and VIEW 2 were phase III, randomized, double- masked, clinical trials (38,39) evaluating VEGF Trap Eye effect on maintaining and improving vision as compared to ranibizumab.The studies have been completed in 2011. VIEW 1 was conducted in USA and VIEW 2 was conducted in Asia, Europe, Japan and Latin America. In both studies patients were randomized evenly to one of four treatment groups:0.5 mg ranibizumab monthly, VEGF trap 0.5 mg monthly, VEGF trap 2 mg monthly or VEGF trap 2 mg dosed every 8

VIEW 1 enrolled 1217 patients. Prevention of moderate vision loss was achieved in 94-96% of patients from all four groups showing VEGF Trap Eye non inferior to ranibizumab. Mean gain in visual acuity in all four groups was as follow: VEGF Trap 0.5 mg group achieved a mean gain of seven letter, the 2 mg VEGF Trap montly group a mean gain of 11 letters, the 2 mg VEGF Trap dosed every two months after initial loading dose gained a mean of 8 letters while ranibizumab monthly group gained a mean of 8 letters. The only statistically significant difference in visual gain was between patients receiving VEGF Trap Eye 2mg monthly compared to ranibizumab monthly with p<0.01 (11 letter vs. 8 letter gain at week

International VIEW 2 study enrolled 1240. As in VIEW 1 study, prevention of moderate vision loss was achieved in 94- 96 percent of patients from all four groups confirming non

A generally favorable safety profile was observed for both VEGF Trap Eye and ranibizumab. The most frequent ocular adverse events were conjunctival hemorrhage,

In conclusion both VIEW 1 and VIEW 2 studies showed VEGF-Trap-Eye being non inferior to monthly ranibizumab in prevention of moderate vision loss with good safety profile and potential for VEGF-Trap to achieve equally superior visual results as monthly ranibizumab with less frequent dosing. When dosed 2 mg monthly VEGF Trap Eye even showed superior

Small interfering RNAs (siRNA) are synthetic nucleotide chains, containing 20 nucleotides. While VEGF antibodies neutralize the already produced VEGF, siRNA interferes with VEGF -A messenger RNA (mRNA)(40,41), inhibiting thereby the production of all VEGF-A

As a result, the already produced VEGF persists within the eye for a couple of the first

SiRNA 027 interferes with the VEGF-R1 mRNA production. As demonstrated by CNV models, intravitreal and periocular injection of siRNA resulted in a significant CNV lesion

injection (37).

weeks following a tree-injection loading dose.

52) showing superiority of VEGF Trap Eye dosed 2mg monthly.

in terms of vision gain compared to ranibizumab monthly

treatment weeks, leading to delayed therapeutic effect.

**4.2 Interfering RNA** 

isoforms.

reduction (42).

inferiority of VEGF Trap Eye at all doses compared to monthly ranibizumab.

macular degeneration, eye pain, retinal hemorrhage, and vitreous floaters.

outcome was maintained (24). Despite its large molecular weight, bevacizumab showed satisfying penetration into subretinal space, without any harmful neurophysiologic effects (25). Many clinical trials confirmed later the efficacy of bevacizumab in improving the visual acuity, reducing the retinal exudation and the acceptable safety profile (26,27,28,29,30).

Bevacizumab and ranibizumab exhibit differences and similarities in the treatment of nAMD:


Several studies showed no difference in efficiency between ranibizumab and bevacizumab(31,32,33). Interesting results came from a study which concluded that functional result were alike, however bevacizumab needed a longer period of time to achieve resolution of retinal edema than ranibizumab: 60 versus 90 days. The period of the drug potency was 110 days for bevacizumab, and 70 days for ranibizumab, suggesting a longer interval of bevacizumab dosing(34).

Comparison of AMD Treatment Trials (CATT) study (35) was designed to compare ranibizumab and bevacizumab in treatment of nAMD. 1208 patients were enrolled. Patients were randomized into four study arms: ranibizumab monthly or as needed and bevacizumab monthly or as needed. Ranibizumab and bevacizumab showed equivalent results in terms of efficiency. Patients treated with monthly bevacizumab gained 8 letters, while patients treated with monthly ranibizumab gained 8.5 letters after 12 month follow-up period. In as-needed arms bevacizumab was equivalent to ranibizumab with 5.9 and 6.8 letters gained.

Ranibizumab as needed was equivalent to monthly ranibizumab, while comparison between bevacizumab as needed and bevacizumab monthly showed inconclusive results. Rates of death, myocardial infarction, and stroke were similar for patients receiving either ranibizumab or bevacizumab. The proportion of patients with serious systemic adverse events (SSAE) requiring hospitalization was higher with bevacizumab than with ranibizumab arms (24.1% vs.19.0%). The importance of the CATT trial was in reporting the same efficacy of bevacizumab in a major randomized clinical trial. The raised concern with higher incidence of SSAE in bevacizumab is worth of noting but still inconclusive and needs to be further analyzed.

#### **4. Drugs under investigation**

#### **4.1 VEGF Trap Eye (aflibercept)**

To initiate molecular mechanism of neovascularization, it is essential for the VEGF to bind to endothelial cell receptors VEGFR1 and VEGFR2, which act as transmembrane tyrosine kinases.

By binding VEGF to their outer subunit, tyrosine kinase is activated which then triggers intracellular signaling pathways. The VEGF Trap Eye is a fusion protein containing VEGFR1

outcome was maintained (24). Despite its large molecular weight, bevacizumab showed satisfying penetration into subretinal space, without any harmful neurophysiologic effects (25). Many clinical trials confirmed later the efficacy of bevacizumab in improving the visual acuity, reducing the retinal exudation and the acceptable safety profile (26,27,28,29,30).

Bevacizumab and ranibizumab exhibit differences and similarities in the treatment of

• Bevacizumab has larger molecular weight, which could interfere with the penetration

• Given that the bevacizumab is a full antibody with the Fc fragment, whereas

Several studies showed no difference in efficiency between ranibizumab and bevacizumab(31,32,33). Interesting results came from a study which concluded that functional result were alike, however bevacizumab needed a longer period of time to achieve resolution of retinal edema than ranibizumab: 60 versus 90 days. The period of the drug potency was 110 days for bevacizumab, and 70 days for ranibizumab, suggesting a

Comparison of AMD Treatment Trials (CATT) study (35) was designed to compare ranibizumab and bevacizumab in treatment of nAMD. 1208 patients were enrolled. Patients were randomized into four study arms: ranibizumab monthly or as needed and bevacizumab monthly or as needed. Ranibizumab and bevacizumab showed equivalent results in terms of efficiency. Patients treated with monthly bevacizumab gained 8 letters, while patients treated with monthly ranibizumab gained 8.5 letters after 12 month follow-up period. In as-needed arms bevacizumab was equivalent to ranibizumab with 5.9 and 6.8

Ranibizumab as needed was equivalent to monthly ranibizumab, while comparison between bevacizumab as needed and bevacizumab monthly showed inconclusive results. Rates of death, myocardial infarction, and stroke were similar for patients receiving either ranibizumab or bevacizumab. The proportion of patients with serious systemic adverse events (SSAE) requiring hospitalization was higher with bevacizumab than with ranibizumab arms (24.1% vs.19.0%). The importance of the CATT trial was in reporting the same efficacy of bevacizumab in a major randomized clinical trial. The raised concern with higher incidence of SSAE in bevacizumab is worth of noting but still inconclusive and needs

To initiate molecular mechanism of neovascularization, it is essential for the VEGF to bind to endothelial cell receptors VEGFR1 and VEGFR2, which act as transmembrane tyrosine

By binding VEGF to their outer subunit, tyrosine kinase is activated which then triggers intracellular signaling pathways. The VEGF Trap Eye is a fusion protein containing VEGFR1

• Bevacizumab has weaker affinity towards the VEGF factor than ranibizumab.

ranibizumab is only an antibody fragment, it might be more immunogenic.

nAMD:

ability.

letters gained.

to be further analyzed.

kinases.

**4. Drugs under investigation 4.1 VEGF Trap Eye (aflibercept)** 

• Both drugs inhibit all VEGF-A isoforms.

longer interval of bevacizumab dosing(34).

and VEGFR2 similar domains combined with Fc immunoglobulin segment (36). This means that VEGF Trap Eye has antibody inhibiting characteristics for all VEGF-A isoforms and binding affinity 800 times greater than bevacizumab. A clinical trial with 25 patients has shown good tolerability and increased visual acuity 6 weeks after single intravitreal injection (37).

VIEW 1 and VIEW 2 were phase III, randomized, double- masked, clinical trials (38,39) evaluating VEGF Trap Eye effect on maintaining and improving vision as compared to ranibizumab.The studies have been completed in 2011. VIEW 1 was conducted in USA and VIEW 2 was conducted in Asia, Europe, Japan and Latin America. In both studies patients were randomized evenly to one of four treatment groups:0.5 mg ranibizumab monthly, VEGF trap 0.5 mg monthly, VEGF trap 2 mg monthly or VEGF trap 2 mg dosed every 8 weeks following a tree-injection loading dose.

VIEW 1 enrolled 1217 patients. Prevention of moderate vision loss was achieved in 94-96% of patients from all four groups showing VEGF Trap Eye non inferior to ranibizumab. Mean gain in visual acuity in all four groups was as follow: VEGF Trap 0.5 mg group achieved a mean gain of seven letter, the 2 mg VEGF Trap montly group a mean gain of 11 letters, the 2 mg VEGF Trap dosed every two months after initial loading dose gained a mean of 8 letters while ranibizumab monthly group gained a mean of 8 letters. The only statistically significant difference in visual gain was between patients receiving VEGF Trap Eye 2mg monthly compared to ranibizumab monthly with p<0.01 (11 letter vs. 8 letter gain at week 52) showing superiority of VEGF Trap Eye dosed 2mg monthly.

International VIEW 2 study enrolled 1240. As in VIEW 1 study, prevention of moderate vision loss was achieved in 94- 96 percent of patients from all four groups confirming non inferiority of VEGF Trap Eye at all doses compared to monthly ranibizumab.

A generally favorable safety profile was observed for both VEGF Trap Eye and ranibizumab. The most frequent ocular adverse events were conjunctival hemorrhage, macular degeneration, eye pain, retinal hemorrhage, and vitreous floaters.

In conclusion both VIEW 1 and VIEW 2 studies showed VEGF-Trap-Eye being non inferior to monthly ranibizumab in prevention of moderate vision loss with good safety profile and potential for VEGF-Trap to achieve equally superior visual results as monthly ranibizumab with less frequent dosing. When dosed 2 mg monthly VEGF Trap Eye even showed superior in terms of vision gain compared to ranibizumab monthly

#### **4.2 Interfering RNA**

Small interfering RNAs (siRNA) are synthetic nucleotide chains, containing 20 nucleotides.

While VEGF antibodies neutralize the already produced VEGF, siRNA interferes with VEGF -A messenger RNA (mRNA)(40,41), inhibiting thereby the production of all VEGF-A isoforms.

As a result, the already produced VEGF persists within the eye for a couple of the first treatment weeks, leading to delayed therapeutic effect.

SiRNA 027 interferes with the VEGF-R1 mRNA production. As demonstrated by CNV models, intravitreal and periocular injection of siRNA resulted in a significant CNV lesion reduction (42).

Treatment of Neovascular Age Related Macular Degeneration 131

vascular tissue by inhibiting neovascularization (51,52). After low-dose radiation, vascular endothelium demonstrates morphologic and DNA changes, inhibition of replication, increased cell permeability, and apoptosis. Fibroblast proliferation and subsequent scar

CNVs which contain proliferating endothelial cells due to the hypoxic environment and the produced chemokines are more sensitive to radiation treatment than the retinal vasculature and non-proliferating capillary endothelial cells and larger vessels. Therefore to reduce complication rate and to improve visual outcome epimacular brachytherapy was introduced. It uses strontium-90 beta radiation as radiation source (NeoVista, Fremont, CA.). Total radiation dose is 24 gy. Epimacular brachytherapy is designed to deliver precisely controlled dose of beta radiation to CNV lesion. Compared to previously used radiation therapy strontium-90 beta radiation is ideal for treating retina because its delivery system ensues no collateral damage to surrounding retinal tissues (53,54). After pars plana vitrectomy is done, radiation applicator is placed directly above CNV lesion and held for 2-4 minutes. This has a dual effect: vitrectomy increases retinal oxygen saturation and in contrast to external beam radiotherapy a larger dose of radiation can be delivered to the macula with less irradiation of normal ocular structures and surrounding tissues. This novel device is currently being evaluated in two prospective, randomized, controlled trials in treatment-naive subjects: the CNV Secondary AMD Treated with Beta Radiation Epretinal Therapy (CABERNET) and in subjects already treated with anti-vascular endothelial growth factor therapy: Macular

Having in mind the multiplicity of signaling mechanisms which are crucial for the development of nAMD as well as multistage evolution of the disease, combination

The anti-VEGF and PDT with verterpofin have up to now been the only available options mostly used in everyday clinical practice. The introduction of the PDT in treatment of nAMD in 2001 for the very first time affected natural course of the disease. This was achieved by acting on the last arm in pathophysiologic cascade of neovascularisation

The second major breakthrough in the therapy of nAMD was the introduction of anti-VEGF

The anti-VEGF drugs act one step earlier in pathogenesis of the disease opposite to PDT, by preventing neovascularization and inducing regression of the newly formed neovascular blood vessels still dependent on VEGF support. Unlike PDT, which could only slow down the disease progression and reduce the visual acuity decline, patients treated with anti-VEGF could expect their visual acuity to be maintained and even improved in significant

Although the anti-VEGF drugs are for the time being the best treatment option in managing nAMD, there are a few setbacks that caused the initial enthusiasm to drop. Regardless of anti-VEGF frequent dosing, a significant number of patients suffer further visual acuity deterioration throughout the course of the disease. Frequent intravitreal applications raise a risk for local complications such as: endophthalmitis, uveitis, vitreous hemorrhage, retinal detachment, posterior vitreous detachment etc. Despite continuous anti-VEGF blockage,

formation, a hallmark of end-stage nAMD are also inhibited.

Epiretinal Brachytherapy versus Lucentis Only Treatment (MERLOT).

process: destruction of already formed neovascular membrane.

treatments could have synergistic effect in halting the progression of disease.

**4.6 Combination treatment** 

drugs.

number of patients.

Other siRNA target hypoxia-induced transcription factor (HIF-1). This factor is important not only in tumor angiogenesis but also in normal vessel formation. HIF-1 is composed of the constitutively expressed HIF-1 beta subunit and the 3-alfa subunit. In non-hypoxia conditions, HIF-1 alpha dissolves rapidly, whereas in the hypoxic environment, HIF-1 alpha becomes stable and acts as a hypoxia-provoked inducible gene regulator (43). Currently there are no ongoing clinical trials investigating this compound in treatment of nAMD but data from previous studies showed the compound to have a potential for VEGF inhibition.

#### **4.3 Tyrosine kinase inhibitors**

One of the most important biochemical mechanisms of intracellular signaling mediation is reverse phosphorilation. This reaction is catalysed by kinase proteins, which transfer gphosphate ATP group to hydroxyl group of targeted proteins (44). There are 518 of such proteins in human genome, 90 of which are selective hydroxyl group tyrosine phosphorilation catalysts(45).

Cytosol tyrosine kinases are intracellular, while the receptor tyrosine kinase (RTK) have intracellular and extracellular domain and function as membrane receptors. The RTKs modulate cellular responses as such to signalling from the environment and act as various cell processes boosters of cellular proliferation, migration and survival. Otherwise the RTK signal mechanisms are well regulated, while their excessive activation can stimulate growth, survival and tumor cell metastasis development (46). Members of the VEGF and PDGF receptor group, which belong to the RTK family, promote tumor progression through various mechanisms: angiogenesis, limphangenesis and vascular permeability.

PTK 787 is a RTK inhibitor with binding affinity to VEGF receptor tyrosine kinase and thus inhibits all VEGF-A isoforms. PTK 787 displayed functional improvement of ischemic retinopathy induced in mice. A single intravitreal injection of PTK reduced angiproliferative changes compared to the control eye of each animal (n=37) when retinopathy scores were compared (47). Currently there are no clinical trials of PTK 787 in treatment of nAMD, but the compound has a potential for VEGF inhibition.

#### **4.4 Cytokine PEDF**

Pigment Epithelial Derived Factor (PEDF), produced by retinal pigment epithelial cells is one of the most important endogenous angiogenesis inhibitors (48). The exact location of the PEDF production is on the apical side of pigment epithelial cells and contrary to VEGF it is inhibited by hypoxia.

A high PEDF concentration can be found in extracellular photoreceptor matter, vitreous and cornea, indicating its major role in maintaining the tissues avascularity (49). The PEDF's anti-angiogenic capacity has been proven in laser-stimulated CNV animal model (50). The PEDF concentrations are lower in the eyes suffering from CNVs, which is consistent with its anti-angiogenic properties. Therefore gene transferring adenovirus coding over-expression of PEDF could suppress the angiogenesis process.

#### **4.5 Epimacular brachytherapy**

Previously used, radiation therapy from external radiation source produced inconsistent results with high rate of side-effects. It was therefore abandoned from everyday clinical practice. Localized radiation treatment, on contrary has an ability to prevent proliferation of

Other siRNA target hypoxia-induced transcription factor (HIF-1). This factor is important not only in tumor angiogenesis but also in normal vessel formation. HIF-1 is composed of the constitutively expressed HIF-1 beta subunit and the 3-alfa subunit. In non-hypoxia conditions, HIF-1 alpha dissolves rapidly, whereas in the hypoxic environment, HIF-1 alpha becomes stable and acts as a hypoxia-provoked inducible gene regulator (43). Currently there are no ongoing clinical trials investigating this compound in treatment of nAMD but data from previous studies showed the compound to have a potential for VEGF inhibition.

One of the most important biochemical mechanisms of intracellular signaling mediation is reverse phosphorilation. This reaction is catalysed by kinase proteins, which transfer gphosphate ATP group to hydroxyl group of targeted proteins (44). There are 518 of such proteins in human genome, 90 of which are selective hydroxyl group tyrosine

Cytosol tyrosine kinases are intracellular, while the receptor tyrosine kinase (RTK) have intracellular and extracellular domain and function as membrane receptors. The RTKs modulate cellular responses as such to signalling from the environment and act as various cell processes boosters of cellular proliferation, migration and survival. Otherwise the RTK signal mechanisms are well regulated, while their excessive activation can stimulate growth, survival and tumor cell metastasis development (46). Members of the VEGF and PDGF receptor group, which belong to the RTK family, promote tumor progression through

PTK 787 is a RTK inhibitor with binding affinity to VEGF receptor tyrosine kinase and thus inhibits all VEGF-A isoforms. PTK 787 displayed functional improvement of ischemic retinopathy induced in mice. A single intravitreal injection of PTK reduced angiproliferative changes compared to the control eye of each animal (n=37) when retinopathy scores were compared (47). Currently there are no clinical trials of PTK 787 in treatment of nAMD, but

Pigment Epithelial Derived Factor (PEDF), produced by retinal pigment epithelial cells is one of the most important endogenous angiogenesis inhibitors (48). The exact location of the PEDF production is on the apical side of pigment epithelial cells and contrary to VEGF it is

A high PEDF concentration can be found in extracellular photoreceptor matter, vitreous and cornea, indicating its major role in maintaining the tissues avascularity (49). The PEDF's anti-angiogenic capacity has been proven in laser-stimulated CNV animal model (50). The PEDF concentrations are lower in the eyes suffering from CNVs, which is consistent with its anti-angiogenic properties. Therefore gene transferring adenovirus coding over-expression

Previously used, radiation therapy from external radiation source produced inconsistent results with high rate of side-effects. It was therefore abandoned from everyday clinical practice. Localized radiation treatment, on contrary has an ability to prevent proliferation of

various mechanisms: angiogenesis, limphangenesis and vascular permeability.

the compound has a potential for VEGF inhibition.

of PEDF could suppress the angiogenesis process.

**4.5 Epimacular brachytherapy** 

**4.3 Tyrosine kinase inhibitors** 

phosphorilation catalysts(45).

**4.4 Cytokine PEDF** 

inhibited by hypoxia.

vascular tissue by inhibiting neovascularization (51,52). After low-dose radiation, vascular endothelium demonstrates morphologic and DNA changes, inhibition of replication, increased cell permeability, and apoptosis. Fibroblast proliferation and subsequent scar formation, a hallmark of end-stage nAMD are also inhibited.

CNVs which contain proliferating endothelial cells due to the hypoxic environment and the produced chemokines are more sensitive to radiation treatment than the retinal vasculature and non-proliferating capillary endothelial cells and larger vessels. Therefore to reduce complication rate and to improve visual outcome epimacular brachytherapy was introduced. It uses strontium-90 beta radiation as radiation source (NeoVista, Fremont, CA.). Total radiation dose is 24 gy. Epimacular brachytherapy is designed to deliver precisely controlled dose of beta radiation to CNV lesion. Compared to previously used radiation therapy strontium-90 beta radiation is ideal for treating retina because its delivery system ensues no collateral damage to surrounding retinal tissues (53,54). After pars plana vitrectomy is done, radiation applicator is placed directly above CNV lesion and held for 2-4 minutes. This has a dual effect: vitrectomy increases retinal oxygen saturation and in contrast to external beam radiotherapy a larger dose of radiation can be delivered to the macula with less irradiation of normal ocular structures and surrounding tissues. This novel device is currently being evaluated in two prospective, randomized, controlled trials in treatment-naive subjects: the CNV Secondary AMD Treated with Beta Radiation Epretinal Therapy (CABERNET) and in subjects already treated with anti-vascular endothelial growth factor therapy: Macular Epiretinal Brachytherapy versus Lucentis Only Treatment (MERLOT).

#### **4.6 Combination treatment**

Having in mind the multiplicity of signaling mechanisms which are crucial for the development of nAMD as well as multistage evolution of the disease, combination treatments could have synergistic effect in halting the progression of disease.

The anti-VEGF and PDT with verterpofin have up to now been the only available options mostly used in everyday clinical practice. The introduction of the PDT in treatment of nAMD in 2001 for the very first time affected natural course of the disease. This was achieved by acting on the last arm in pathophysiologic cascade of neovascularisation process: destruction of already formed neovascular membrane.

The second major breakthrough in the therapy of nAMD was the introduction of anti-VEGF drugs.

The anti-VEGF drugs act one step earlier in pathogenesis of the disease opposite to PDT, by preventing neovascularization and inducing regression of the newly formed neovascular blood vessels still dependent on VEGF support. Unlike PDT, which could only slow down the disease progression and reduce the visual acuity decline, patients treated with anti-VEGF could expect their visual acuity to be maintained and even improved in significant number of patients.

Although the anti-VEGF drugs are for the time being the best treatment option in managing nAMD, there are a few setbacks that caused the initial enthusiasm to drop. Regardless of anti-VEGF frequent dosing, a significant number of patients suffer further visual acuity deterioration throughout the course of the disease. Frequent intravitreal applications raise a risk for local complications such as: endophthalmitis, uveitis, vitreous hemorrhage, retinal detachment, posterior vitreous detachment etc. Despite continuous anti-VEGF blockage,

Treatment of Neovascular Age Related Macular Degeneration 133

The RADICAL was a phase II, multicentric, randomized, single-masked study of 162 patients with nAMD. The purpose of the study was to determine whether the PDT combined with ranibizumab reduced re-treatment rate compared with ranibizumab monotherapy. Patients were randomized into 4 groups: ranibizumab monotherapy, triple therapy with quarterfluence verteporfin followed by ranibizumab and then dexamethasone, triple therapy with half-fluence verteporfin followed by ranibizumab and then dexamethasone and double therapy with half-fluence verteporfin followed by ranibizumab. The 24-month results showed significantly fewer retreatments in combination groups than in ranibizumab monotherapy group. Mean visual acuity change was not statistically different among the treatment groups. Through 24 months, patients in the triple therapy half-fluence group had a mean of 4.2 retreatment visits compared with 8.9 for patients who received ranibizumab monotherapy. At the month 24, mean VA in the triple therapy half-fluence group improved 1.8 letters fewer compared with the ranibizumab monotherapy group which was not significantly inferior. This results show a potential of combination therapy in reducing the retreatment rate while sustaining the same visual outcome. The concept of combination therapy with new emerging

drugs could further show a synergistic effect when those drugs would be combined.

year follow-up period.

showing exudation and hemorrhage.

The displayed figures depict 2 patients from an extension of our pilot study throughout the period of 3 years. Both patients were treatment naïve. First patient N.U. was randomized to bevacizumab monotherapy treatment group and was treated with bevacizumab only and second patient R.K. was randomized to combination treatment group and was treated with combination treatment initially and then bevacizumab as needed. The second patient treated with combination treatment required less intravitreal bevacizumab injections during a 3

Fig. 1. Patient N.U. 76 yrs., fundus photography at baseline before bevacizumab treatment

active signaling pathways and expression of VEGF genes lead to continuous VEGF production, so continuous and regular treatment over a longer period of time is required as anti-VEGF drugs block only already produced VEGF.

Besides already mentioned increased complication risks, another problem is cost of anti-VEGF drugs as well as discomfort caused by intravitreal mode of application which ultimately leads to poor patient compliance. Having this in mind, new treatment options should be investigated. A potential new therapy regimen should actually have the following characteristics:


In pursuit of better treatment modality in 2006 we proposed a combination of PDT and anti-VEGF with an idea to address two different steps of CNV formation: VEGF induced neovascularization and destruction of already formed CNV. In future a third potential drug acting on gene transcription could also be included thus preventing revascularization even earlier in the cascade of events. Further experimental and clinical trials are needed to support this hypothesis.

Clinical studies conducted between 2001 and 2006, i.e. before the introduction of anti-VEGF drugs, investigated a possibility of a combination treatment for nAMD using PDT and trimacinolone. Trimacinolone is a long-acting corticosteroid, otherwise employed in treatment of rheumatic diseases of locomotor system. In ophthalmology trimacinolone was used intravitreally for diabetic macular edema. A significant number of studies demonstrated the same functional outcome of triamcinolone and PDT combined versus the PDT alone, with longer remission interval and a reduced need for additional reapplications of PDT when combined with triamcinolone(55,56,57,58). Since the combination therapy of triamcinolone and PDT proved to have synergistic effect, we hypothesized that combination of anti-VEGF drugs and PDT could either improve functional outcome or extend treatment intervals in patients with nAMD. We suggested a possible synergistic effect due to different target-points of choroidal neovascularization process: PDT inducing vascular occlusion to already formed neovascular vessels while anti-VEGF drugs preventing formation of new neovascular tissue, inducing regression of the newly formed VEGF-dependent vessels and reducing permeability of neovascular tissue. Additional production of VEGF after PDTinduced hypoxia of choriocapilaris and inflammatory reaction due to neovascular tissue destruction could also be targeted with anti-VEGF drugs. In 2006, we concluded a pilot study on a small number of patients, divided into three groups - one treated with PDT, one with bevacizumab and the last one with bevacizumab and PDT together. The achieved results indicated possible synergistic effect: the visual acuity was better in patients who underwent combination therapy than in monotherapy groups, whereas the remission period was longer in combination group (59).

Other studies also indicated possible amplifying effect of the combination treatment with PDT and anti-VEGF. Also some studies included addition of intravitreal corticosteroid to address the inflammatory component of the disease (60-70).

active signaling pathways and expression of VEGF genes lead to continuous VEGF production, so continuous and regular treatment over a longer period of time is required as

Besides already mentioned increased complication risks, another problem is cost of anti-VEGF drugs as well as discomfort caused by intravitreal mode of application which ultimately leads to poor patient compliance. Having this in mind, new treatment options should be investigated. A potential new therapy regimen should actually have the following

In pursuit of better treatment modality in 2006 we proposed a combination of PDT and anti-VEGF with an idea to address two different steps of CNV formation: VEGF induced neovascularization and destruction of already formed CNV. In future a third potential drug acting on gene transcription could also be included thus preventing revascularization even earlier in the cascade of events. Further experimental and clinical trials are needed to

Clinical studies conducted between 2001 and 2006, i.e. before the introduction of anti-VEGF drugs, investigated a possibility of a combination treatment for nAMD using PDT and trimacinolone. Trimacinolone is a long-acting corticosteroid, otherwise employed in treatment of rheumatic diseases of locomotor system. In ophthalmology trimacinolone was used intravitreally for diabetic macular edema. A significant number of studies demonstrated the same functional outcome of triamcinolone and PDT combined versus the PDT alone, with longer remission interval and a reduced need for additional reapplications of PDT when combined with triamcinolone(55,56,57,58). Since the combination therapy of triamcinolone and PDT proved to have synergistic effect, we hypothesized that combination of anti-VEGF drugs and PDT could either improve functional outcome or extend treatment intervals in patients with nAMD. We suggested a possible synergistic effect due to different target-points of choroidal neovascularization process: PDT inducing vascular occlusion to already formed neovascular vessels while anti-VEGF drugs preventing formation of new neovascular tissue, inducing regression of the newly formed VEGF-dependent vessels and reducing permeability of neovascular tissue. Additional production of VEGF after PDTinduced hypoxia of choriocapilaris and inflammatory reaction due to neovascular tissue destruction could also be targeted with anti-VEGF drugs. In 2006, we concluded a pilot study on a small number of patients, divided into three groups - one treated with PDT, one with bevacizumab and the last one with bevacizumab and PDT together. The achieved results indicated possible synergistic effect: the visual acuity was better in patients who underwent combination therapy than in monotherapy groups, whereas the remission period

Other studies also indicated possible amplifying effect of the combination treatment with PDT and anti-VEGF. Also some studies included addition of intravitreal corticosteroid to

anti-VEGF drugs block only already produced VEGF.

characteristics:

5. Acceptable cost.

support this hypothesis.

1. Increased treatment efficacy. 2. Prolonged remission period. 3. Low reapplication rate. 4. Low complications rate.

6. Comfortable mode of administration.

was longer in combination group (59).

address the inflammatory component of the disease (60-70).

The RADICAL was a phase II, multicentric, randomized, single-masked study of 162 patients with nAMD. The purpose of the study was to determine whether the PDT combined with ranibizumab reduced re-treatment rate compared with ranibizumab monotherapy. Patients were randomized into 4 groups: ranibizumab monotherapy, triple therapy with quarterfluence verteporfin followed by ranibizumab and then dexamethasone, triple therapy with half-fluence verteporfin followed by ranibizumab and then dexamethasone and double therapy with half-fluence verteporfin followed by ranibizumab. The 24-month results showed significantly fewer retreatments in combination groups than in ranibizumab monotherapy group. Mean visual acuity change was not statistically different among the treatment groups. Through 24 months, patients in the triple therapy half-fluence group had a mean of 4.2 retreatment visits compared with 8.9 for patients who received ranibizumab monotherapy. At the month 24, mean VA in the triple therapy half-fluence group improved 1.8 letters fewer compared with the ranibizumab monotherapy group which was not significantly inferior. This results show a potential of combination therapy in reducing the retreatment rate while sustaining the same visual outcome. The concept of combination therapy with new emerging drugs could further show a synergistic effect when those drugs would be combined.

The displayed figures depict 2 patients from an extension of our pilot study throughout the period of 3 years. Both patients were treatment naïve. First patient N.U. was randomized to bevacizumab monotherapy treatment group and was treated with bevacizumab only and second patient R.K. was randomized to combination treatment group and was treated with combination treatment initially and then bevacizumab as needed. The second patient treated with combination treatment required less intravitreal bevacizumab injections during a 3 year follow-up period.

Fig. 1. Patient N.U. 76 yrs., fundus photography at baseline before bevacizumab treatment showing exudation and hemorrhage.

Treatment of Neovascular Age Related Macular Degeneration 135

Fig. 4. Patient N.U. 76 yrs, OCT scan (Optopol SD device). It shows the resolution of fluid 3

Fig. 5. Patient R.K. 74 yrs., fundus photography at baseline before combination treatment

(bevacizumab + PDT) showing exudation and hemorrhage.

years after repeated intravitreal bevacizumab treatment.

Fig. 2. Patient N.U. 76 yrs, early and late phase fluorescein angiography pictures at baseline before bevacizumab treatment showing blockage of retrofluorescence by hemorrhage, and a leakage of dye from the CNV.

Fig. 3. Patient N.U. 76 yrs OCT scan (Zeiss Stratus II device) at baseline before bevacizumab treatment. It shows accumulation of intraretinal and subepithelial fluid.

Fig. 2. Patient N.U. 76 yrs, early and late phase fluorescein angiography pictures at baseline before bevacizumab treatment showing blockage of retrofluorescence by hemorrhage, and a

Fig. 3. Patient N.U. 76 yrs OCT scan (Zeiss Stratus II device) at baseline before bevacizumab

treatment. It shows accumulation of intraretinal and subepithelial fluid.

leakage of dye from the CNV.

Fig. 4. Patient N.U. 76 yrs, OCT scan (Optopol SD device). It shows the resolution of fluid 3 years after repeated intravitreal bevacizumab treatment.

Fig. 5. Patient R.K. 74 yrs., fundus photography at baseline before combination treatment (bevacizumab + PDT) showing exudation and hemorrhage.

Treatment of Neovascular Age Related Macular Degeneration 137

Fig. 8. Patient R.K. 74 yrs OCT scan (Optopol SD device). It shows resolution of fluid 3 years after initial combination treatment of intravitreal bevacizumab and photodynamic therapy

In summary we can conclude that new emerging therapies for nAMD for the first time in history managed to revert the natural history of disease. In significant number of patients some improvement could be achieved while in a majority of patients the treatment resulted in maintenance of visual acuity. However the significant burden of repeated intravitreal injections, increased risks of ocular and possibly systemic side effects and decreased patients' compliance lead to further visual loss over time. Also some patients do not respond to the available treatments favorably. So the need for new and more efficient drugs in terms of better functional outcome and reduced need for retreatment is fully justified. VEGF-Trap-Eye is pending approval and it may show to be more potent and requiring less treatment. Also combination of present treatment modalities should further be evaluated. Hopefully with better understanding of the genes responsible for different variants of nAMD we could either employ some form of genetic therapy or we can adjust already available treatments according to a certain genotype in order to achieve most favorable

[1] Macular Photocoagulation Study Group. Argon laser photocoagulation for neovascular

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**5. References** 

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Fig. 6. Patient R.K. 74 yrs, early and late phase of fluorescein angiography at baseline before combination treatment (bevacizumab+PDT). It shows marginal hemorrhage and leakage of dye from the CNV.

Fig. 7. Patient R.K. 74 yrs OCT scan (Zeiss Stratus II device) at baseline before combination treatment. It shows accumulation of intraretinal and subretinal fluid.

Fig. 6. Patient R.K. 74 yrs, early and late phase of fluorescein angiography at baseline before combination treatment (bevacizumab+PDT). It shows marginal hemorrhage and leakage of

Fig. 7. Patient R.K. 74 yrs OCT scan (Zeiss Stratus II device) at baseline before combination

treatment. It shows accumulation of intraretinal and subretinal fluid.

dye from the CNV.

Fig. 8. Patient R.K. 74 yrs OCT scan (Optopol SD device). It shows resolution of fluid 3 years after initial combination treatment of intravitreal bevacizumab and photodynamic therapy followed by repeated bevacizumab treatment.

In summary we can conclude that new emerging therapies for nAMD for the first time in history managed to revert the natural history of disease. In significant number of patients some improvement could be achieved while in a majority of patients the treatment resulted in maintenance of visual acuity. However the significant burden of repeated intravitreal injections, increased risks of ocular and possibly systemic side effects and decreased patients' compliance lead to further visual loss over time. Also some patients do not respond to the available treatments favorably. So the need for new and more efficient drugs in terms of better functional outcome and reduced need for retreatment is fully justified. VEGF-Trap-Eye is pending approval and it may show to be more potent and requiring less treatment. Also combination of present treatment modalities should further be evaluated. Hopefully with better understanding of the genes responsible for different variants of nAMD we could either employ some form of genetic therapy or we can adjust already available treatments according to a certain genotype in order to achieve most favorable results.

#### **5. References**


Treatment of Neovascular Age Related Macular Degeneration 139

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**8** 

*Turkey* 

**Re-Treatment Strategies for Neovascular** 

*1Gazi University, School of Medicine, Department of Ophthalmology, Ankara 2Yenisehir State Hospital, Department of Ophthalmology, Kahramanmaras* 

Age-related macular degeneration (AMD) is a leading cause of severe, irreversible vision impairment in developed countries (Friedman et al., 2004; Klein et al., 1992). Although an estimated 80% of patients with AMD have the non-neovascular form, the neovascular form is responsible for almost 90% of severe visual loss (visual acuity 20/200 or worse) resulting from AMD (Ferris et al., 1984). There was no effective treatment for most of the neovascular AMD lesions till 2004. By this time, the role of vascular endothelial growth factor (VEGF) in neovascular AMD became obvious and anti-VEGF agents emerged for this purpose. Pegaptanib sodium intravitreal injection (Macugen; [OSI] Eyetech, New York, NY, 2004), ranibizumab intravitreal injection (Lucentis, Genentech, Inc., South San Francisco, CA, 2006) were the FDA approved treatments for AMD. The first report of intravitreal bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) administration for neovascular AMD was published in 2005 (Rosenfeld et al., 2005). By early 2006, off-label intravitreal bevacizumab was used by many retina specialists as a first-line therapy for neovascular AMD because of the low cost of this drug. Wholesale prices of the medications range from \$1950 per dose for ranibizumab and \$995 per dose for pegaptanib, to approximately \$50 per

MARINA and ANCHOR were the first studies showing level 1 evidence for the effect of ranibizumab for the treatment of neovascular ARMD (Brown et al., 2009; Rosenfeld et al., 2006a; Rosenfeld et al., 2006b). These studies have shown that 33-40% of the eyes with neovascular AMD treated with ranibizumab gained 15 letters or more (Brown et al., 2009; Rosenfeld et al., 2006a). However these studies used monthly injections of the drug for 24 months. This is the most effective treatment but almost impossible to apply in routine applications both for patients and doctors. Additionally monthly injections will cost so much that treatment cannot be afforded neither by patients nor the social security systems.

**2. How to decrease the number of injections without compromising visual** 

PIER study was the first study investigating the results of less frequent dosing regimens with ranibizumab (3-monthly) for the treatment of AMD (Regillo et al., 2008). However

dose for bevacizumab (Champan & Beckey, 2006; Web, 2008).

**1. Introduction** 

**acuity?** 

**AMD: When to Treat? When to Stop?** 

Sengul Ozdek1 and Mehmet Cuneyt Ozmen2


### **Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop?**

Sengul Ozdek1 and Mehmet Cuneyt Ozmen2 *1Gazi University, School of Medicine, Department of Ophthalmology, Ankara 2Yenisehir State Hospital, Department of Ophthalmology, Kahramanmaras Turkey* 

#### **1. Introduction**

142 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

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and photodynamic therapy to treat exudative age-related macular degeneration.

combined with intravitreal bevacizumab for neovascular age-related macular

verteporfin and ranibizumab; optimizing the timing in the CAM model.

Age-related macular degeneration (AMD) is a leading cause of severe, irreversible vision impairment in developed countries (Friedman et al., 2004; Klein et al., 1992). Although an estimated 80% of patients with AMD have the non-neovascular form, the neovascular form is responsible for almost 90% of severe visual loss (visual acuity 20/200 or worse) resulting from AMD (Ferris et al., 1984). There was no effective treatment for most of the neovascular AMD lesions till 2004. By this time, the role of vascular endothelial growth factor (VEGF) in neovascular AMD became obvious and anti-VEGF agents emerged for this purpose. Pegaptanib sodium intravitreal injection (Macugen; [OSI] Eyetech, New York, NY, 2004), ranibizumab intravitreal injection (Lucentis, Genentech, Inc., South San Francisco, CA, 2006) were the FDA approved treatments for AMD. The first report of intravitreal bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) administration for neovascular AMD was published in 2005 (Rosenfeld et al., 2005). By early 2006, off-label intravitreal bevacizumab was used by many retina specialists as a first-line therapy for neovascular AMD because of the low cost of this drug. Wholesale prices of the medications range from \$1950 per dose for ranibizumab and \$995 per dose for pegaptanib, to approximately \$50 per dose for bevacizumab (Champan & Beckey, 2006; Web, 2008).

MARINA and ANCHOR were the first studies showing level 1 evidence for the effect of ranibizumab for the treatment of neovascular ARMD (Brown et al., 2009; Rosenfeld et al., 2006a; Rosenfeld et al., 2006b). These studies have shown that 33-40% of the eyes with neovascular AMD treated with ranibizumab gained 15 letters or more (Brown et al., 2009; Rosenfeld et al., 2006a). However these studies used monthly injections of the drug for 24 months. This is the most effective treatment but almost impossible to apply in routine applications both for patients and doctors. Additionally monthly injections will cost so much that treatment cannot be afforded neither by patients nor the social security systems.

#### **2. How to decrease the number of injections without compromising visual acuity?**

PIER study was the first study investigating the results of less frequent dosing regimens with ranibizumab (3-monthly) for the treatment of AMD (Regillo et al., 2008). However

Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 145

Although PRN treatment regimen may reduce the number of intravitreal injections and allow the treatment plan to be individualized, it may still require monthly visits to specialized centers. In contrast to mandated monthly injections, patients treated with PRN strategies may develop multiple recurrences of CNV activity over time. Recurrent intra- or subretinal fluid could potentially induce progressive, cumulative dysfunction of the neural retina, resulting in a decreased ability of the retina to recover despite further treatment. There are some other treatment regimens like "treat and extend" and "individualized injection intervals" regimens which aim to individualize the treatment plan and decrease the number of injections per year, but at the same time attempts to achieve a fluid-free macula and decrease the number of visits (Brown & Regillo, 2007; Gupta et al., 2010; Hörster et al.,

In an attempt to minimize the number of intravitreal injections, office visits, and ancillary testing, a "treat and extend" regimen (TER) was first put forth by Bailey Freund, (unpublished data, February 2006) and then adopted by others (Gupta et al., 2010; Oubraham et al., 2011). A typical TER starts with monthly injections until the signs of exudation have resolved with confirmation by OCT. The treatment interval is then sequentially lengthened by 1 to 2 weeks as long as there are no signs of recurrent exudation. When recurrent exudation is detected on a follow-up visit, the treatment interval is reduced to the prior interval. Treatment is rendered at every visit but the time between visits is individualized based on a given patient's response to treatment. As with traditional PRN regimens, the goal is to maintain an exudation-free macula with the fewest number of injections. This approach also may allow for a significant reduction in

In a study by Gupta et al., eyes with neovascular AMD experienced significant visual improvement when managed with intravitreal ranibizumab using a TER. This treatment approach also was associated with significantly fewer patient visits, injections, and direct annual medical cost compared with monthly injections such as in the phase III clinical trials (Gupta et al., 2010). The interval was individualized for each patient in an attempt to maintain an exudation-free macula. In another study comparing the results of this treatment regimen with the standard PRN regimen, patients reinjected by the TER had a far better visual outcome than PRN regimen but needed more injections (Oubraham et al., 2011).

Another individualized treatment strategy that aims to avoid recurrent CNV activity in addition to reducing the number of injections and visits may be to perform the injection immediately prior to the next recurrence. This would require the ability to determine or predict the recurrence interval for an individual patient. A treatment schedule can be obtained for some of the cases after a couple of years of experience with PRN regimen (Hörster et al., 2011). Knowledge of individual recurrence interval times may allow for the

**2.2 Treatment regimens other than PRN** 

2011; Oubraham et al., 2011; Spaide, 2007).

**2.2.1 Treat & extend dosing regimen** 

**2.2.2 Individualized injection intervals** 

development of an individualized treatment plan (Figure 1).

office visits and tests.

PIER study revealed disappointing results showing loss of early gained vision during 3 monthly injection period. Other prospective studies, PrONTO and SUSTAIN, investigated the efficacy of PRN (pro re nata; as needed) treatment regimen. Results from these studies suggested that fewer injections by using a variable dosing regimen with OCT will most likely result in visual acuity improvements similar to the results from the phase III trials which used monthly injections (Holz et al., 2011; Lalwani et al., 2009; Rosenfeld et al., 2006b)

#### **2.1 What is "PRN" treatment regimen and what are the most reliable criteria for treatment decision?**

PRN treatment regimen is a treatment schedule which allows treatment only if the lesion is active. The aim of the PRN treatment is to avoid monthly injections and to decrease the number of injections as much as possible while preserving the vision gained in the loading period (first three months).

The key point in PRN treatment is assessment of the activity of the lesion to decide for additional treatments. It is obvious that a totally fibrotic yellow scarring lesion without any hemorrhage around, only late staining of the scar tissue in fluorescein angiography (FA), and no subretinal or intraretinal fluid in optical coherence tomography (OCT) with stable vision for a long time does not need any treatment. On the other side of the spectrum, a lesion with subretinal hemorrhages all around, significant late leakage in FA and a considerable subretinal or intraretinal fluid in OCT and deteriorating vision recently needs treatment with no doubt. However most of the lesions are in between these two ends of the spectrum especially during the course of anti-VEGF treatments and may be difficult to decide if any further treatment is necessary or not.

Until recently, the presence or absence of fluorescein leakage and the angiographic appearance of the lesion were the main criteria for the decision to treat neovascular ARMD and re-treat using PDT or anti-VEGF therapy (Schmidt-Erfurth et al., 2007). Within the last decade OCT has emerged and enhanced our understanding in many central retinal diseases. For AMD, OCT appears to be useful for evaluating the responses of the retina and retinal pigment epithelium (RPE) to the treatment (Cohen et al., 2007; Eter & Spaide, 2005; Fung et al., 2007; Krebs et al., 2008; Lalwani et al., 2009; Salinas-Alamán et al., 2005; Schmidt-Erfurth et al., 2007; van Velthoven et al., 2006;). Clinical trials have shown that in the two thirds of patients requiring ranibizumab therapy because of recurrent neovascularization, OCT seemed to detect early anatomic changes in the macula before any vision loss (observation during the extension trials of phase I-II studies by P. Rosenfeld: unpublished data). The PrONTO study was initiated to explore the use of OCT as the basis for a less frequent variable dosing regimen with ranibizumab (Fung et al., 2007; Lalwani et al., 2009). This study used some criteria for retreatment including an OCT based parameter (an increase in central OCT thickness of at least 100 mm) during the first year. However they made an amendment to their OCT based criteria in the second year. They changed the retreatment criteria to include any qualitative change in the appearance of the OCT images that suggested recurrent fluid in the macula. These qualitative changes included the appearance of retinal cysts or subretinal fluid or an enlargement of a PED. Any of these qualitative changes alone was sufficient to permit retreatment (Lalwani et al., 2009).

#### **2.2 Treatment regimens other than PRN**

144 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

PIER study revealed disappointing results showing loss of early gained vision during 3 monthly injection period. Other prospective studies, PrONTO and SUSTAIN, investigated the efficacy of PRN (pro re nata; as needed) treatment regimen. Results from these studies suggested that fewer injections by using a variable dosing regimen with OCT will most likely result in visual acuity improvements similar to the results from the phase III trials which used monthly injections (Holz et al., 2011; Lalwani et al., 2009; Rosenfeld et al., 2006b)

**2.1 What is "PRN" treatment regimen and what are the most reliable criteria for** 

PRN treatment regimen is a treatment schedule which allows treatment only if the lesion is active. The aim of the PRN treatment is to avoid monthly injections and to decrease the number of injections as much as possible while preserving the vision gained in the loading

The key point in PRN treatment is assessment of the activity of the lesion to decide for additional treatments. It is obvious that a totally fibrotic yellow scarring lesion without any hemorrhage around, only late staining of the scar tissue in fluorescein angiography (FA), and no subretinal or intraretinal fluid in optical coherence tomography (OCT) with stable vision for a long time does not need any treatment. On the other side of the spectrum, a lesion with subretinal hemorrhages all around, significant late leakage in FA and a considerable subretinal or intraretinal fluid in OCT and deteriorating vision recently needs treatment with no doubt. However most of the lesions are in between these two ends of the spectrum especially during the course of anti-VEGF treatments and may be difficult to

Until recently, the presence or absence of fluorescein leakage and the angiographic appearance of the lesion were the main criteria for the decision to treat neovascular ARMD and re-treat using PDT or anti-VEGF therapy (Schmidt-Erfurth et al., 2007). Within the last decade OCT has emerged and enhanced our understanding in many central retinal diseases. For AMD, OCT appears to be useful for evaluating the responses of the retina and retinal pigment epithelium (RPE) to the treatment (Cohen et al., 2007; Eter & Spaide, 2005; Fung et al., 2007; Krebs et al., 2008; Lalwani et al., 2009; Salinas-Alamán et al., 2005; Schmidt-Erfurth et al., 2007; van Velthoven et al., 2006;). Clinical trials have shown that in the two thirds of patients requiring ranibizumab therapy because of recurrent neovascularization, OCT seemed to detect early anatomic changes in the macula before any vision loss (observation during the extension trials of phase I-II studies by P. Rosenfeld: unpublished data). The PrONTO study was initiated to explore the use of OCT as the basis for a less frequent variable dosing regimen with ranibizumab (Fung et al., 2007; Lalwani et al., 2009). This study used some criteria for retreatment including an OCT based parameter (an increase in central OCT thickness of at least 100 mm) during the first year. However they made an amendment to their OCT based criteria in the second year. They changed the retreatment criteria to include any qualitative change in the appearance of the OCT images that suggested recurrent fluid in the macula. These qualitative changes included the appearance of retinal cysts or subretinal fluid or an enlargement of a PED. Any of these qualitative changes alone was sufficient to permit retreatment (Lalwani et

**treatment decision?** 

al., 2009).

period (first three months).

decide if any further treatment is necessary or not.

Although PRN treatment regimen may reduce the number of intravitreal injections and allow the treatment plan to be individualized, it may still require monthly visits to specialized centers. In contrast to mandated monthly injections, patients treated with PRN strategies may develop multiple recurrences of CNV activity over time. Recurrent intra- or subretinal fluid could potentially induce progressive, cumulative dysfunction of the neural retina, resulting in a decreased ability of the retina to recover despite further treatment. There are some other treatment regimens like "treat and extend" and "individualized injection intervals" regimens which aim to individualize the treatment plan and decrease the number of injections per year, but at the same time attempts to achieve a fluid-free macula and decrease the number of visits (Brown & Regillo, 2007; Gupta et al., 2010; Hörster et al., 2011; Oubraham et al., 2011; Spaide, 2007).

#### **2.2.1 Treat & extend dosing regimen**

In an attempt to minimize the number of intravitreal injections, office visits, and ancillary testing, a "treat and extend" regimen (TER) was first put forth by Bailey Freund, (unpublished data, February 2006) and then adopted by others (Gupta et al., 2010; Oubraham et al., 2011). A typical TER starts with monthly injections until the signs of exudation have resolved with confirmation by OCT. The treatment interval is then sequentially lengthened by 1 to 2 weeks as long as there are no signs of recurrent exudation. When recurrent exudation is detected on a follow-up visit, the treatment interval is reduced to the prior interval. Treatment is rendered at every visit but the time between visits is individualized based on a given patient's response to treatment. As with traditional PRN regimens, the goal is to maintain an exudation-free macula with the fewest number of injections. This approach also may allow for a significant reduction in office visits and tests.

In a study by Gupta et al., eyes with neovascular AMD experienced significant visual improvement when managed with intravitreal ranibizumab using a TER. This treatment approach also was associated with significantly fewer patient visits, injections, and direct annual medical cost compared with monthly injections such as in the phase III clinical trials (Gupta et al., 2010). The interval was individualized for each patient in an attempt to maintain an exudation-free macula. In another study comparing the results of this treatment regimen with the standard PRN regimen, patients reinjected by the TER had a far better visual outcome than PRN regimen but needed more injections (Oubraham et al., 2011).

#### **2.2.2 Individualized injection intervals**

Another individualized treatment strategy that aims to avoid recurrent CNV activity in addition to reducing the number of injections and visits may be to perform the injection immediately prior to the next recurrence. This would require the ability to determine or predict the recurrence interval for an individual patient. A treatment schedule can be obtained for some of the cases after a couple of years of experience with PRN regimen (Hörster et al., 2011). Knowledge of individual recurrence interval times may allow for the development of an individualized treatment plan (Figure 1).

Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 147

In conclusion, re-injections of ranibizumab shortly prior to a recurrence may avoid recurrent leakage and fluid accumulation, as well as further growth of the CNV lesion size. Therefore, avoiding recurrent CNV activity with prophylactic injections may protect the neural retina from additional damage and improve the long-term prognosis. However, this approach requires the ability to determine individual recurrence intervals with a couple of years of

All treatment regimens mentioned above aims to reduce injection numbers without compromising the patients visual acuity. Yet there is not a consensus about the objective

Definition of an active lesion should not be done only with the OCT based criteria. We believe that, other parameters like visual acuity, presence of hemorrhage associated with the lesion, lesion size and FA staining pattern (when needed) are all also important for assessment of neovascular AMD activity. The PrONTO study put some of these parameters together and created their criteria for retreatment. Retreatment with ranibizumab was

SUSTAIN study used only VA and OCT criteria for retreatment decision. We believe that these are very well prepared criteria, however, some of the items could be changed and

A new clinical activity scoring (AS) is proposed to assess activity of lesions, to quantify the activity for statistical purposes in clinical studies and to standardize the re-treatment protocols during the course of anti-VEGF treatments of neovascular AMD lesions. This may

The proposed AS is based on the well known and widely used signs and findings of active

performed only if one of the following occurred in PrONTO study (Fung et al., 2007):

**2.3 The need for an activity scoring for re-treatment of exudative AMD** 

experience with PRN regimen.

criteria of an active neovascular CNV lesion.

3. New-onset classic neovascularization,

4. New macular hemorrhage.

be a basis for treatment regimens.

neovascular AMD (Table 1):

4. Change in size of the lesion,

5. FA staining pattern (when needed).

3. Change in vision;

**3.1 Methods** 

1. An increase in central OCT thickness of at least 100 mm,

some new criteria could be added to make it more reliable.

1. Presence of subretinal or intraretinal fluid in OCT, 2. Presence of hemorrhage associated with the lesion,

a. Objective measured visual acuity (VA)

b. Subjective vision (what patient feels about his vision)

2. A loss of five letters in conjunction with recurrent fluid by OCT,

**3. A clinical activity scoring for re-treatment of exudative AMD** 

Fig. 1. A, Baseline, VA: 44 letters, 1st injection. B, 1st month, 53 letters, 2nd injection. C, 2nd month, 54 letters, 3rd injection. D, 51 letters, no treatment. E, 4th month, 46 letters, 4th injection. F, 5th month, 47 letters, no treatment. G, 6th month, 46 letters, 5th injection. H, 7th month, 42 letters, 6th injection. I, 8th month, 50 letters, no treatment. J, 8th month, 44 letters, 7th injection. K, 9th month, 48 letters, no treatment. L, 10th month, 38 letters, 8th injection. According to the data, this patient has a recurrence pattern of 8 weeks, thus needs retreatment every 7 weeks.

A retrospective study in University of Cologne analyzed the recurrence intervals of patients undergoing anti-VEGF therapy for neovascular AMD to determine whether predictable, regular recurrence patterns were present for individual patients (Hörster et al., 2011). The paper reported that, all recurrences occurred at regular intervals in 41% of the eyes and the recurrence interval time may vary between individuals (Hörster et al., 2011).

In conclusion, re-injections of ranibizumab shortly prior to a recurrence may avoid recurrent leakage and fluid accumulation, as well as further growth of the CNV lesion size. Therefore, avoiding recurrent CNV activity with prophylactic injections may protect the neural retina from additional damage and improve the long-term prognosis. However, this approach requires the ability to determine individual recurrence intervals with a couple of years of experience with PRN regimen.

#### **2.3 The need for an activity scoring for re-treatment of exudative AMD**

All treatment regimens mentioned above aims to reduce injection numbers without compromising the patients visual acuity. Yet there is not a consensus about the objective criteria of an active neovascular CNV lesion.

Definition of an active lesion should not be done only with the OCT based criteria. We believe that, other parameters like visual acuity, presence of hemorrhage associated with the lesion, lesion size and FA staining pattern (when needed) are all also important for assessment of neovascular AMD activity. The PrONTO study put some of these parameters together and created their criteria for retreatment. Retreatment with ranibizumab was performed only if one of the following occurred in PrONTO study (Fung et al., 2007):


SUSTAIN study used only VA and OCT criteria for retreatment decision. We believe that these are very well prepared criteria, however, some of the items could be changed and some new criteria could be added to make it more reliable.

#### **3. A clinical activity scoring for re-treatment of exudative AMD**

A new clinical activity scoring (AS) is proposed to assess activity of lesions, to quantify the activity for statistical purposes in clinical studies and to standardize the re-treatment protocols during the course of anti-VEGF treatments of neovascular AMD lesions. This may be a basis for treatment regimens.

#### **3.1 Methods**

146 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Fig. 1. A, Baseline, VA: 44 letters, 1st injection. B, 1st month, 53 letters, 2nd injection. C, 2nd month, 54 letters, 3rd injection. D, 51 letters, no treatment. E, 4th month, 46 letters, 4th injection. F, 5th month, 47 letters, no treatment. G, 6th month, 46 letters, 5th injection. H, 7th month, 42 letters, 6th injection. I, 8th month, 50 letters, no treatment. J, 8th month, 44 letters, 7th injection. K, 9th month, 48 letters, no treatment. L, 10th month, 38 letters, 8th injection. According to the data, this patient has a recurrence pattern of 8 weeks, thus needs re-

A retrospective study in University of Cologne analyzed the recurrence intervals of patients undergoing anti-VEGF therapy for neovascular AMD to determine whether predictable, regular recurrence patterns were present for individual patients (Hörster et al., 2011). The paper reported that, all recurrences occurred at regular intervals in 41% of the eyes and the

recurrence interval time may vary between individuals (Hörster et al., 2011).

treatment every 7 weeks.

The proposed AS is based on the well known and widely used signs and findings of active neovascular AMD (Table 1):

	- a. Objective measured visual acuity (VA)
	- b. Subjective vision (what patient feels about his vision)


Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 149

2. **Amount of hemorrhage**: The amount of hemorrhage associated with the lesion (in ophthalmoscopy, colored fundus photography or FA) was noted and if there is any hemorrhage at the beginning it was scored as 2. If there is no hemorrhage it was scored as 0, if it was decreased it was scored as 1, if it was the same, scored as 2, and if it was

a. *Objective VA* is measured with ETDRS and noted as a baseline and scored as 1. If there is a decrease in vision (any line or ≥5 letters loss) it was scored as 2 and if

there is any increase in vision (any line or ≥5 letters gain) it was scored as 0. b. *Subjective vision* (what patient feels about his vision): Patient's feeling about any change in his vision was also asked and noted as subjective vision which was scored as 0 if he feels better, 2 if he feels worse and 1 if he did not feel any change

4. **FA staining pattern**: No staining or window defect (0), staining of scar tissue or PED (1)

5. **The area of the lesion** (mm2): It was measured in FA and the baseline area (or no change) was scored as 1, at least 10% (of the original area) increase was scored as 2 and

Records of the patients at the 1st month visit after the treatment were also noted and all of the above parameters were again noted, so that an AS is calculated both before and after the treatment. AS could range between 0 and 14. Change in AS after the treatment was analyzed

It can be hypothesized that, the more active the lesion the more it may respond to the anti-VEGF therapy. A possible correlation between pretreatment AS and posttreatment decrease

Eyes with favorable treatment response (two or more units of decrease in AS) and unfavorable treatment response (one unit or no decrease in AS) were separated and the mean pre and posttreatment AS were calculated for both groups. The mean AS of these two groups were compared by using Mann-Whitney U test. At this stage, a cut off point for AS was searched to determine the eyes that need treatment. Sensitivity, specificity, negative and positive predictive values of the AS were calculated to determine the cut off point for AS

In this section, the results of an ongoing study is presented to better understand the activity

A total of 52 eyes with neovascular AMD were involved in the study. Mean age of the patients was 72.7 (52-89), mean visual acuity (logMAR) was 0.68 (0-1.6) and mean lesion

Pretreatment mean AS of eyes was 7.4 (ranged between 3 and 10) which decreased significantly to 4.2 after treatment (p<0.001, Wilcoxon). There was a significant positive correlation between the pretreatment AS of eyes and the posttreatment decrease in AS

in AS was investigated by using Pearson correlation test to test this hypothesis.

increased scored as 3. 3. Visual assessment:

(baseline).

and late leakage (2) were noted.

by using Wilcoxon signed rank test.

at least 10% decrease was scored as 0.

which will be used to decide on re-treatments.

area was 8.9 mm2 (0.6-33mm2) before treatment.

(Pearson correlation coefficient: 0.534, p<0.001, Figure 2).

**3.2 Results** 

scoring.


Table 1. Clinical Activity Scoring for neovascular AMD lesions

Apart from FA staining pattern, all of the other assessments are based on the changes (samebaseline/increased/decreased) in each parameter and given a number to define the activity. At the end of the assessment, given numbers are summed and an activity score is calculated.

This scoring has been used in a group of neovascular AMD patients all of which have been involved in a prospective study for intravitreal bevacizumab (IVB) in our clinic (Şekeryapan et al., 2011). All of them received IVB monotherapy. The reports of the patients were reviewed retrospectively and demographic features as well as lesion characteristics of the patients were noted. AS of all of the lesions were calculated according to the following criteria:

1. **OCT:** OCT was performed using the Humphrey model 3000 (Zeiss-Humphrey Instruments, San Leandro, CA). After pupil dilatation, six consecutive 6mm long scans containing 128 axial profiles (A-scans) at equally spaced angular orientations in a radial spoke pattern centered on the fovea (known as Fast Macular Thickness Protocol) were obtained for each eye. Using Retinal Thickness Mapping Software mean retinal thickness value which was measured in the central disc with a diameter of 1000µm in the center of the macula was used as central foveal thickness (CFT). The fluid pattern (subretinal / intraretinal diffuse / cystoid / pigment epithelial detachment - PED) was also noted. Only the CFT was used as an activity parameter in AS and at least 10% increase or decrease in CFT was accepted as a decrease or increase. The amount of fluid at the beginning was scored as 2. It was scored as "0" if there is no fluid, "1" if there is a decrease and "3 if there is an increase in CFT.


No hemorrhage 0 decreased 1 Same amount 2 increased 3

None 0 Decreased 1 Any amount at beginning / Stable 2 Increased 3

Increased 0 No change 1 Decreased 2

Increased 0 No change 1 Decreased 2

No staining/window defect 0 Staining of scar tissue/PED 1 Late leakage 2

Decreased 0 Beginning size / Stable 1 Increased 2

Apart from FA staining pattern, all of the other assessments are based on the changes (samebaseline/increased/decreased) in each parameter and given a number to define the activity. At the end of the assessment, given numbers are summed and an activity score is calculated. This scoring has been used in a group of neovascular AMD patients all of which have been involved in a prospective study for intravitreal bevacizumab (IVB) in our clinic (Şekeryapan et al., 2011). All of them received IVB monotherapy. The reports of the patients were reviewed retrospectively and demographic features as well as lesion characteristics of the patients were noted. AS of all of the lesions were calculated according to the following

1. **OCT:** OCT was performed using the Humphrey model 3000 (Zeiss-Humphrey Instruments, San Leandro, CA). After pupil dilatation, six consecutive 6mm long scans containing 128 axial profiles (A-scans) at equally spaced angular orientations in a radial spoke pattern centered on the fovea (known as Fast Macular Thickness Protocol) were obtained for each eye. Using Retinal Thickness Mapping Software mean retinal thickness value which was measured in the central disc with a diameter of 1000µm in the center of the macula was used as central foveal thickness (CFT). The fluid pattern (subretinal / intraretinal diffuse / cystoid / pigment epithelial detachment - PED) was also noted. Only the CFT was used as an activity parameter in AS and at least 10% increase or decrease in CFT was accepted as a decrease or increase. The amount of fluid at the beginning was scored as 2. It was scored as "0" if there is no fluid, "1" if there is a

**PARAMETER GRADING SCORE**

Table 1. Clinical Activity Scoring for neovascular AMD lesions

decrease and "3 if there is an increase in CFT.

**Hemorrhage** 

**OCT** 

Amount of hemorrhage associated with the lesion

Subretinal fluid / retinal thickening / PED

**Visual assessment** 

**Visual assessment** 

Staining pattern

**Size of the lesion**  Lesion area in FA

**Objective**

**Subjective**

**FA** 

criteria:


Records of the patients at the 1st month visit after the treatment were also noted and all of the above parameters were again noted, so that an AS is calculated both before and after the treatment. AS could range between 0 and 14. Change in AS after the treatment was analyzed by using Wilcoxon signed rank test.

It can be hypothesized that, the more active the lesion the more it may respond to the anti-VEGF therapy. A possible correlation between pretreatment AS and posttreatment decrease in AS was investigated by using Pearson correlation test to test this hypothesis.

Eyes with favorable treatment response (two or more units of decrease in AS) and unfavorable treatment response (one unit or no decrease in AS) were separated and the mean pre and posttreatment AS were calculated for both groups. The mean AS of these two groups were compared by using Mann-Whitney U test. At this stage, a cut off point for AS was searched to determine the eyes that need treatment. Sensitivity, specificity, negative and positive predictive values of the AS were calculated to determine the cut off point for AS which will be used to decide on re-treatments.

#### **3.2 Results**

In this section, the results of an ongoing study is presented to better understand the activity scoring.

A total of 52 eyes with neovascular AMD were involved in the study. Mean age of the patients was 72.7 (52-89), mean visual acuity (logMAR) was 0.68 (0-1.6) and mean lesion area was 8.9 mm2 (0.6-33mm2) before treatment.

Pretreatment mean AS of eyes was 7.4 (ranged between 3 and 10) which decreased significantly to 4.2 after treatment (p<0.001, Wilcoxon). There was a significant positive correlation between the pretreatment AS of eyes and the posttreatment decrease in AS (Pearson correlation coefficient: 0.534, p<0.001, Figure 2).

Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 151

decrease was statistically significant (p<0.001, Figure 4). The mean pretreatment AS was 5.4 (3-6) in group 2 which decreased to 4 (1-6) after treatment. Although the decrease was less than that in group 1, it was still statistically significant (p=0.034, Figure 4). The mean

Groups

Favorable Response Unfavorable Response Total

Fig. 3. The pretreatment AS was statistically significantly higher in favorable treatment response group than unfavorable treatment response group (Mann Whitney U test p=0.003).

was 45% (table 3). These may be assumed as indicators for accuracy of AS.

AS ≥7 37 4 41 AS <7 6 5 11 Total 43 9 52 Positive predictive value, 37 of 41 = 90%; negative predictive value, 5 of 11 = 45%, sensitivity,

Table 3. Accuracy of activity score (AS) in predicting therapeutic outcome of treatment in

Sensitivity of AS (ratio of eyes with a favorable response and an AS of 7 or more to the total number of eyes with favorable response) was 86% and, specificity of AS (ratio of eyes with an unfavorable response and an AS of less than 7 to the total number of eyes with unfavorable response) was 56% with a cut-off point of 7 (table 3). Positive predictive value (ratio of eyes with AS of 7 or more and a favorable response to the total number of eyes with AS of 7 or more) of AS was 90% and negative predictive value (ratio of eyes with AS of less than 7 and an unfavorable response to the total number of eyes with AS of less than 7) of AS

Favorable response Unfavorable response

decrease in AS was 3.5 in group one and 1.4 in group two (p=0.003, Figure 5).

Mean Pretreatment AS +/- 2 SE

8,5

8,0

7,5

7,0

6,5

6,0

5,5

5,0

4,5

37 of 43 = 86%, specificity, 5 of 9 = 56%.

neovascular AMD.

Difference in AS (Pre-Post)

Fig. 2. Correlation between the pretreatment AS of eyes and the post-treatment decrease in AS (Pearson correlation coefficient: 0.534, p<0.001)

To define a cut-off point a group of eyes with favorable treatment responses is formed by separating those who had at least 2 point decrease in AS and named the favorable response group. The remaining eyes formed the unfavorable response group. The pretreatment mean AS was 7.5 (6-10) in favorable response group and 6 (3-7) in unfavorable response group. The pretreatment AS in favorable treatment response group was statistically significantly higher than those of unfavorable treatment response groups (p=0.003, Figure 3).

The sensitivity, specificity, negative and positive predictive values of the AS with different cut off points were calculated (table 2) and an AS of 7 was found to be most suitable as a cutoff point for further analysis.


Table 2. Predictive values, sensitivity and specificity of AS for detecting favorable treatment response (2 or more decrease in AS)

Eyes with an AS of 7 or more (group 1, highly active group, n=41) were separated from those less than 7 (group 2, less active group, n=11) and a subgroup analysis was done. The mean AS in group 1 was 7.8 (7-10) before treatment and 4.3 (1-10) after treatment. The


Fig. 2. Correlation between the pretreatment AS of eyes and the post-treatment decrease in

To define a cut-off point a group of eyes with favorable treatment responses is formed by separating those who had at least 2 point decrease in AS and named the favorable response group. The remaining eyes formed the unfavorable response group. The pretreatment mean AS was 7.5 (6-10) in favorable response group and 6 (3-7) in unfavorable response group. The pretreatment AS in favorable treatment response group was statistically significantly

The sensitivity, specificity, negative and positive predictive values of the AS with different cut off points were calculated (table 2) and an AS of 7 was found to be most suitable as a cut-

> Pretreatment AS≥ 7

Pretreatment AS≥ 8

higher than those of unfavorable treatment response groups (p=0.003, Figure 3).

Pretreatment AS≥ 6

Positive predictive value 85,10% 90,20% 100,00% Negative predictive value 40,00% 45,50% 25,70% Sensitivity 93,00% 86,00% 39,50% Specificity 22,20% 55,60% 100,00%

Table 2. Predictive values, sensitivity and specificity of AS for detecting favorable treatment

Eyes with an AS of 7 or more (group 1, highly active group, n=41) were separated from those less than 7 (group 2, less active group, n=11) and a subgroup analysis was done. The mean AS in group 1 was 7.8 (7-10) before treatment and 4.3 (1-10) after treatment. The

Difference in AS (Pre-Post)

Pretreatment AS

off point for further analysis.

response (2 or more decrease in AS)

12

10

8

6

4

2

AS (Pearson correlation coefficient: 0.534, p<0.001)

decrease was statistically significant (p<0.001, Figure 4). The mean pretreatment AS was 5.4 (3-6) in group 2 which decreased to 4 (1-6) after treatment. Although the decrease was less than that in group 1, it was still statistically significant (p=0.034, Figure 4). The mean decrease in AS was 3.5 in group one and 1.4 in group two (p=0.003, Figure 5).

Fig. 3. The pretreatment AS was statistically significantly higher in favorable treatment response group than unfavorable treatment response group (Mann Whitney U test p=0.003).

Sensitivity of AS (ratio of eyes with a favorable response and an AS of 7 or more to the total number of eyes with favorable response) was 86% and, specificity of AS (ratio of eyes with an unfavorable response and an AS of less than 7 to the total number of eyes with unfavorable response) was 56% with a cut-off point of 7 (table 3). Positive predictive value (ratio of eyes with AS of 7 or more and a favorable response to the total number of eyes with AS of 7 or more) of AS was 90% and negative predictive value (ratio of eyes with AS of less than 7 and an unfavorable response to the total number of eyes with AS of less than 7) of AS was 45% (table 3). These may be assumed as indicators for accuracy of AS.


Positive predictive value, 37 of 41 = 90%; negative predictive value, 5 of 11 = 45%, sensitivity, 37 of 43 = 86%, specificity, 5 of 9 = 56%.

Table 3. Accuracy of activity score (AS) in predicting therapeutic outcome of treatment in neovascular AMD.

Cut-off AS

Fig. 4. Pretreatment and posttreatment mean AS of eyes in group 1 (with an AS of 7 or more, highly active group) and in group 2 (with an AS of less than 7, less active group).

Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 153

Neovascular AMD activity is an important factor to determine if it should be treated or not. It is important not to over-treat these eyes to avoid from injection and drug related complications as well as high cost of the treatment. There is no standard protocol for the treatment and retreatment schedule of these eyes and most ophthalmologists use their clinical experience to decide. Following the disappointing results from the quarterly regimen of the PIER study, which showed us loss of early gained vision during 3-monthly injection period (Regillo et al., 2008), new prospective studies like PrONTO, and SUSTAIN are under way investigating less frequent dosing regimens and preliminary results from these studies suggest that fewer injections (mean: 9.9 injections within 24 months in PrONTO, 5.7 injections within 12 months in SUSTAIN) will most likely result in visual acuity improvements similar to the results from the phase III trials by using a variable dosing regimen with OCT (Fung et al., 2007; Lalwani et al., 2009; Rosenfeld et al.,

Although most of the studies used OCT findings and VA changes to determine the need for additional therapy (Holz et al., 2011), we believe that, some other parameters like, subjective feeling of patients about their vision, presence of hemorrhage associated with the lesion, lesion size, FA staining pattern and are also important for assessment of neovascular AMD activity especially in those undetermined cases. AS is defined to standardize the understanding of findings and definition of active lesion. We are studying on this system since 2002 and have used it in our practice as well as in some of our studies (Ozdek et

It is obvious that ophthalmoscopic appearance of a lesion is very important during interpretation of OCT and FA findings. This is to see new subretinal hemorrhages, exudates and fibrotic scar tissues so that FA and OCT findings can correctly be interpreted. OCT is a very important indicator of neovascular AMD activity and may be assumed as the main determinant for deciding the need for re-treatment in patients with AMD. Recently, there is a tendency to assess the activity of neovascular AMD lesions with only OCT without performing FA after the initial assessment (Brown & Regillo, 2007; Cohen et al., 2007; Rosenfeld et al., 2006b; Salinas-Alamán et al., 2005). This approach has emerged especially after anti-VEGF treatments of neovascular AMD lesions to avoid from monthly FA. Once the diagnosis of neovascular AMD was established with FA before treatment, OCT was reported to have a sensitivity of 96% for detecting lesion activity and a diagnostic efficiency (proportion of correct results) of 83% (Salinas-Alamán et al., 2005). However, OCT cannot detect other features including blood, lipid, and vascular patterns so effectively. When a subretinal new hemorrhage associated with the lesion appears in an eye with a dry OCT and FA (without any late leakage) most of the retina specialists assume it as an active lesion and treat it. This is the case when activation takes place on a far edge of the subfoveal neovascular AMD lesion. Salinas et al reported that, OCT may be complementary to FA, especially in cases in which FA was inconclusive. They have found that OCT was considered as positive (presence of sub-retinal fluid and/or intraretinal fluid) in 14.2% of the cases in which FA did not show clear leakage. On the other hand, most of the cases with positive OCT findings without any leakage on FA (false positives) had a disciform scar with persistent cystic cavities on OCT (Salinas-Alamán et al., 2005). It can be concluded that if there is no fibrotic scar visible during the fundus examination, the presence of remaining

**4. Discussion** 

2006b).

al.,2005; Ozdek et al.,2007).

Cut-off AS

Fig. 5. Pre and post treatment difference in AS of highly active group and less active group. Decrease in AS was significantly more in highly active group (pretreatment AS≥7) (p=0.003).

#### **4. Discussion**

152 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Cut-off AS

highly active group) and in group 2 (with an AS of less than 7, less active group).

Fig. 4. Pretreatment and posttreatment mean AS of eyes in group 1 (with an AS of 7 or more,

Cut-off AS

Fig. 5. Pre and post treatment difference in AS of highly active group and less active group. Decrease in AS was significantly more in highly active group (pretreatment AS≥7) (p=0.003).

Pretreatment AS >=7 Pretreatment AS <7

Pretreatment AS >=7 Pretreatment AS <7

Pretreatment AS

Posttreatment AS

Mean Activity Score +/- 2 SE

Difference in AS (Pretreatment - Posttreatment)

9

8

7

6

5

4

3

2

8

6

4

2

0



Neovascular AMD activity is an important factor to determine if it should be treated or not. It is important not to over-treat these eyes to avoid from injection and drug related complications as well as high cost of the treatment. There is no standard protocol for the treatment and retreatment schedule of these eyes and most ophthalmologists use their clinical experience to decide. Following the disappointing results from the quarterly regimen of the PIER study, which showed us loss of early gained vision during 3-monthly injection period (Regillo et al., 2008), new prospective studies like PrONTO, and SUSTAIN are under way investigating less frequent dosing regimens and preliminary results from these studies suggest that fewer injections (mean: 9.9 injections within 24 months in PrONTO, 5.7 injections within 12 months in SUSTAIN) will most likely result in visual acuity improvements similar to the results from the phase III trials by using a variable dosing regimen with OCT (Fung et al., 2007; Lalwani et al., 2009; Rosenfeld et al., 2006b).

Although most of the studies used OCT findings and VA changes to determine the need for additional therapy (Holz et al., 2011), we believe that, some other parameters like, subjective feeling of patients about their vision, presence of hemorrhage associated with the lesion, lesion size, FA staining pattern and are also important for assessment of neovascular AMD activity especially in those undetermined cases. AS is defined to standardize the understanding of findings and definition of active lesion. We are studying on this system since 2002 and have used it in our practice as well as in some of our studies (Ozdek et al.,2005; Ozdek et al.,2007).

It is obvious that ophthalmoscopic appearance of a lesion is very important during interpretation of OCT and FA findings. This is to see new subretinal hemorrhages, exudates and fibrotic scar tissues so that FA and OCT findings can correctly be interpreted. OCT is a very important indicator of neovascular AMD activity and may be assumed as the main determinant for deciding the need for re-treatment in patients with AMD. Recently, there is a tendency to assess the activity of neovascular AMD lesions with only OCT without performing FA after the initial assessment (Brown & Regillo, 2007; Cohen et al., 2007; Rosenfeld et al., 2006b; Salinas-Alamán et al., 2005). This approach has emerged especially after anti-VEGF treatments of neovascular AMD lesions to avoid from monthly FA. Once the diagnosis of neovascular AMD was established with FA before treatment, OCT was reported to have a sensitivity of 96% for detecting lesion activity and a diagnostic efficiency (proportion of correct results) of 83% (Salinas-Alamán et al., 2005). However, OCT cannot detect other features including blood, lipid, and vascular patterns so effectively. When a subretinal new hemorrhage associated with the lesion appears in an eye with a dry OCT and FA (without any late leakage) most of the retina specialists assume it as an active lesion and treat it. This is the case when activation takes place on a far edge of the subfoveal neovascular AMD lesion. Salinas et al reported that, OCT may be complementary to FA, especially in cases in which FA was inconclusive. They have found that OCT was considered as positive (presence of sub-retinal fluid and/or intraretinal fluid) in 14.2% of the cases in which FA did not show clear leakage. On the other hand, most of the cases with positive OCT findings without any leakage on FA (false positives) had a disciform scar with persistent cystic cavities on OCT (Salinas-Alamán et al., 2005). It can be concluded that if there is no fibrotic scar visible during the fundus examination, the presence of remaining

Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 155

Vision is not only the central VA and it has many other components like scotomas in visual field, contrast sensitivity, color vision etc. So snellen or ETDRS visual acuity measurement may not be enough to assess vision especially in macular diseases. The simplest measure may be to ask the patient his feelings about his vision; if it is the same, decreased or increased. The subjective change in vision may also give important clues about the effect of treatment. However, it is highly dependent on the patients' personality and on the eye (in the better eye or in the worse eye). Patient may feel always worse if he is pessimistic and depressive or vice versa. Patient may feel the changes more precisely if the problem is in the better eye, on the other hand, may not feel the significant changes if the disease is in the worse eye. To overcome such shortcomings of subjective assessment of vision, more objective measures for the assessment of the other components of the central vision may be used. VFQ25 may be an option but takes a long time for these cases and is not so practical. Unver et al have developed a new tool for the automated assessment of functional central vision called central field acuity perimetry to solve such problems (Unver et al., 2009). However there is no clinical study with central field acuity perimetry for this purpose up till now. Microperimetry and functional magnetic resonance imaging are other new tools to measure objective measure of topographic visual function (Baseler et al., 2011; Uppal et al., 2011). On the other hand, deterioration of vision is not always an indication of lesion activity itself. Progression of the dry component of the disease (atrophic changes), fibrosis and cicatrisation of the lesion during healing period following treatment may also cause deterioration of vision. Vision cannot be a sole criterion (just like other parameters) but may add to other factors indicating

Change in lesion area is another important determinant of the activity of the lesion. It is not seldom to see a central inactive lesion without any fluid in OCT or any change in objective measured VA may enlarge with a pseudo-pod like extension from one side indicating an active lesion. They usually feel this difference as a subjective worsening of the vision. On the other side, the lesion area may become smaller after treatment indicating a decreased activity of the lesion. This is also an important complementary parameter for assessment of lesion activity and the response to treatment that needs to be taken into account during

The major problem that we faced during the assessment of the reliability of this scoring system was the absence of a gold standard to define an active lesion which can be used for comparison. Rosenfeld et al were the first to define some criteria to identify an active lesion which needs retreatment with anti-VEGF agents in PrONTO study (Ozdek et al., 2005; Rosenfeld et al., 2006b). However, these criteria were not tested for reliability or sensitivity. Our scoring system has some differences from the criteria used in PrONTO study. Firstly, we used a 10% change in CFT in OCT to be significant instead of 100 micron increase for all cases. Sometimes only 30 microns of increase in CFT may be an important change (especially in minimally edematous fovea) on the other hand, even 100 microns of increase in OCT may be meaningless (especially in highly edematous/elevated fovea). Secondly, we have added change in size of the lesion as another criterion. Thirdly, we used subjective changes in vision as another criterion to decide on retreatments. This is because most of the patients feel some very early changes in lesion activity before any change become apparent

lesion activity.

assessment especially in those gray cases.

fluid on the OCT scan may indicate residual lesion activity. It is also valid to assume that a hyperreflective structure on OCT could be misinterpreted without a fundus examination. Therefore, we believe that OCT, FA, and a fundus examination are complementary examinations that should be interpreted together in those undetermined cases.

Although we used FA as a parameter in AS, we do not mean to say that we have to perform FA at every visit. Actually, we need to score the lesion only if it is not so clear that the lesion is active or not what we call as "undetermined cases"(Figure 6) . In other words, if we are not sure that a patient should be retreated or not, we can apply to the AS just to bring all the parameters together. If we still do not want to perform a FA, we can add only 1 point for FA which is neutral for activity scoring for FA.

Fig. 6. 85 year old woman with a neovascular AMD on the left eye received four doses of intravitreal bevacizumab. On the last visit she had lost 5 letters with a subjective visual impairment. A, early phase of the angiogram. B, late phase of the angiogram. C, fundus photograph. D, OCT image of fovea. This would be an undetermined case without FA. Although the OCT has no sign of active lesion, there are late leakage in FA and objective and subjective visual loss. According to table 1 the patient has an activity score of 8 and assessed as an active lesion.

Change in VA is another very important determinant factor in assessment of treatment effect on neovascular AMD. Usually worsening of VA is a sign of bad response to the treatment and, a stable or increased VA is supposed as a favorable treatment response. It is possible to see patients with a dry OCT and silent ophthalmoscopy without any hemorrhage having a decreased VA both objectively and subjectively. Those patients may have a late leakage in FA indicating activity and treatment need or that VA decrease may be a sign of progression of dry component of AMD.

fluid on the OCT scan may indicate residual lesion activity. It is also valid to assume that a hyperreflective structure on OCT could be misinterpreted without a fundus examination. Therefore, we believe that OCT, FA, and a fundus examination are complementary

Although we used FA as a parameter in AS, we do not mean to say that we have to perform FA at every visit. Actually, we need to score the lesion only if it is not so clear that the lesion is active or not what we call as "undetermined cases"(Figure 6) . In other words, if we are not sure that a patient should be retreated or not, we can apply to the AS just to bring all the parameters together. If we still do not want to perform a FA, we can add only 1 point for FA

Fig. 6. 85 year old woman with a neovascular AMD on the left eye received four doses of intravitreal bevacizumab. On the last visit she had lost 5 letters with a subjective visual impairment. A, early phase of the angiogram. B, late phase of the angiogram. C, fundus photograph. D, OCT image of fovea. This would be an undetermined case without FA. Although the OCT has no sign of active lesion, there are late leakage in FA and objective and subjective visual loss. According to table 1 the patient has an activity score of 8 and assessed

Change in VA is another very important determinant factor in assessment of treatment effect on neovascular AMD. Usually worsening of VA is a sign of bad response to the treatment and, a stable or increased VA is supposed as a favorable treatment response. It is possible to see patients with a dry OCT and silent ophthalmoscopy without any hemorrhage having a decreased VA both objectively and subjectively. Those patients may have a late leakage in FA indicating activity and treatment need or that VA decrease may be a sign of

examinations that should be interpreted together in those undetermined cases.

which is neutral for activity scoring for FA.

as an active lesion.

progression of dry component of AMD.

Vision is not only the central VA and it has many other components like scotomas in visual field, contrast sensitivity, color vision etc. So snellen or ETDRS visual acuity measurement may not be enough to assess vision especially in macular diseases. The simplest measure may be to ask the patient his feelings about his vision; if it is the same, decreased or increased. The subjective change in vision may also give important clues about the effect of treatment. However, it is highly dependent on the patients' personality and on the eye (in the better eye or in the worse eye). Patient may feel always worse if he is pessimistic and depressive or vice versa. Patient may feel the changes more precisely if the problem is in the better eye, on the other hand, may not feel the significant changes if the disease is in the worse eye. To overcome such shortcomings of subjective assessment of vision, more objective measures for the assessment of the other components of the central vision may be used. VFQ25 may be an option but takes a long time for these cases and is not so practical. Unver et al have developed a new tool for the automated assessment of functional central vision called central field acuity perimetry to solve such problems (Unver et al., 2009). However there is no clinical study with central field acuity perimetry for this purpose up till now. Microperimetry and functional magnetic resonance imaging are other new tools to measure objective measure of topographic visual function (Baseler et al., 2011; Uppal et al., 2011). On the other hand, deterioration of vision is not always an indication of lesion activity itself. Progression of the dry component of the disease (atrophic changes), fibrosis and cicatrisation of the lesion during healing period following treatment may also cause deterioration of vision. Vision cannot be a sole criterion (just like other parameters) but may add to other factors indicating lesion activity.

Change in lesion area is another important determinant of the activity of the lesion. It is not seldom to see a central inactive lesion without any fluid in OCT or any change in objective measured VA may enlarge with a pseudo-pod like extension from one side indicating an active lesion. They usually feel this difference as a subjective worsening of the vision. On the other side, the lesion area may become smaller after treatment indicating a decreased activity of the lesion. This is also an important complementary parameter for assessment of lesion activity and the response to treatment that needs to be taken into account during assessment especially in those gray cases.

The major problem that we faced during the assessment of the reliability of this scoring system was the absence of a gold standard to define an active lesion which can be used for comparison. Rosenfeld et al were the first to define some criteria to identify an active lesion which needs retreatment with anti-VEGF agents in PrONTO study (Ozdek et al., 2005; Rosenfeld et al., 2006b). However, these criteria were not tested for reliability or sensitivity. Our scoring system has some differences from the criteria used in PrONTO study. Firstly, we used a 10% change in CFT in OCT to be significant instead of 100 micron increase for all cases. Sometimes only 30 microns of increase in CFT may be an important change (especially in minimally edematous fovea) on the other hand, even 100 microns of increase in OCT may be meaningless (especially in highly edematous/elevated fovea). Secondly, we have added change in size of the lesion as another criterion. Thirdly, we used subjective changes in vision as another criterion to decide on retreatments. This is because most of the patients feel some very early changes in lesion activity before any change become apparent

Re-Treatment Strategies for Neovascular AMD: When to Treat? When to Stop? 157

individual recurrence intervals with a couple of years of experience with PRN regimen and some lesions do not obey any rule of periodical recurrence in long term. The results of these approaches need to be proven with further randomized controlled studies to be

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in FA, OCT or ETDRS visual acuity testing. We strongly believe that this should be taken into account during assessment of lesion activity.

We would like to emphasize on the unequal distribution of points between different parameters of the AS. We have purposefully given higher scores for hemorrhage (3 points), OCT (3 points) and VA (2+2 points) which are more powerful indicators of the lesion activity than the FA and lesion size.

When we take all of these parameters into account and score it, we observed that pretreatment mean AS was 7.4 which decreased significantly to 4.2 after treatment. Which means that AS really indicates the activity of the lesion. We also observed that a lesion with a higher AS is more likely to give more dramatic response to the anti-VEGF treatment with a more significant decrease in AS (Figure 4).

Transferring these data to the clinical applications, it seems logical to treat lesions with an AS of 7 or more with anti-VEGF therapy. The high sensitivity (86%) and positive predictive values (90%) of the AS strongly suggest retreatment of lesions with an AS of 7 or more. However the lower sensitivity and negative predictive values of AS weakly supports observation of the ones with lower AS without treatment. The lower rates are most possibly because of the lower number of the eyes with less AS who had still been treated with anti-VEGF. When the number of such eyes had been equal to the treated eyes with higher AS, the specificity and negative predictive values of AS might have been higher which would make the AS a more reliable measure.

In addition to routine clinical practice, AS may be used as a standard way of assessment of lesion activity especially in clinical studies for the statistical comparison of the results. AS may be a valuable tool to see the picture (both the lesion and the response to the treatment) as a whole.

#### **5. Conclusions**

In conclusion, assessment of the lesion activity is important for PRN treatment approaches and AS seems to be a standardized measure to assess the activity of the lesion at the beginning as well as the treatment effect after anti-VEGF therapy. It may be modified with use of some other tools like central field acuity perimetry to be more objective. A lesion with an AS of 7 or more seems to be an active lesion which needs treatment and it most possibly will give a favorable response to anti-VEGF treatment as a decrease in activity. However, the sensitivity and specificity of AS needs to be tested with further studies with larger number of patients to be conclusive. Additionally scoring of the lesion activity quantifies the lesion activity allowing for statistical comparisons between different treatment methods in clinical studies.

Individualized approaches, on the other side, may be a good option in suitable cases. Reinjections shortly prior to a recurrence may avoid further growth of the CNV lesion size, protecting the neural retina from additional damage and improve the long-term prognosis. This may be a better option than PRN approaches preventing recurrences other than treating the recurrence. However, this approach requires the ability to determine individual recurrence intervals with a couple of years of experience with PRN regimen and some lesions do not obey any rule of periodical recurrence in long term. The results of these approaches need to be proven with further randomized controlled studies to be conclusive.

#### **6. References**

156 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

in FA, OCT or ETDRS visual acuity testing. We strongly believe that this should be taken

We would like to emphasize on the unequal distribution of points between different parameters of the AS. We have purposefully given higher scores for hemorrhage (3 points), OCT (3 points) and VA (2+2 points) which are more powerful indicators of the lesion

When we take all of these parameters into account and score it, we observed that pretreatment mean AS was 7.4 which decreased significantly to 4.2 after treatment. Which means that AS really indicates the activity of the lesion. We also observed that a lesion with a higher AS is more likely to give more dramatic response to the anti-VEGF treatment with a

Transferring these data to the clinical applications, it seems logical to treat lesions with an AS of 7 or more with anti-VEGF therapy. The high sensitivity (86%) and positive predictive values (90%) of the AS strongly suggest retreatment of lesions with an AS of 7 or more. However the lower sensitivity and negative predictive values of AS weakly supports observation of the ones with lower AS without treatment. The lower rates are most possibly because of the lower number of the eyes with less AS who had still been treated with anti-VEGF. When the number of such eyes had been equal to the treated eyes with higher AS, the specificity and negative predictive values of AS might have been higher which would make

In addition to routine clinical practice, AS may be used as a standard way of assessment of lesion activity especially in clinical studies for the statistical comparison of the results. AS may be a valuable tool to see the picture (both the lesion and the response to the treatment)

In conclusion, assessment of the lesion activity is important for PRN treatment approaches and AS seems to be a standardized measure to assess the activity of the lesion at the beginning as well as the treatment effect after anti-VEGF therapy. It may be modified with use of some other tools like central field acuity perimetry to be more objective. A lesion with an AS of 7 or more seems to be an active lesion which needs treatment and it most possibly will give a favorable response to anti-VEGF treatment as a decrease in activity. However, the sensitivity and specificity of AS needs to be tested with further studies with larger number of patients to be conclusive. Additionally scoring of the lesion activity quantifies the lesion activity allowing for statistical comparisons between different treatment methods in

Individualized approaches, on the other side, may be a good option in suitable cases. Reinjections shortly prior to a recurrence may avoid further growth of the CNV lesion size, protecting the neural retina from additional damage and improve the long-term prognosis. This may be a better option than PRN approaches preventing recurrences other than treating the recurrence. However, this approach requires the ability to determine

into account during assessment of lesion activity.

activity than the FA and lesion size.

more significant decrease in AS (Figure 4).

the AS a more reliable measure.

as a whole.

**5. Conclusions** 

clinical studies.


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A & Staurenghi G, SUSTAIN Study Group, (2011). Safety and efficacy of a flexible dosing regimen of ranibizumab in neovascular age-related macular degeneration: the SUSTAIN study. *Ophthalmology,* Vol.118, No.4, (April 2011), pp.663-71, ISSN

anti-VEGF therapy for age-related macular degeneration. *Graefes Arch Clin Exp* 

Dam Eye Study. *Ophthalmology*, Vol.99, No.6, (June 1992), pp.933–43, ISSN 0161-

treated with bevacizumab: comparison between optical coherence tomography and fluorescein angiography. *Graefes Arch Clin Exp Ophthalmol.,* Vol.246, No.6, (June

Flynn HW Jr & Esquiabro M, (2009). A variable-dosing regimen with intravitreal ranibizumab for neovascular age-related macular degeneration: year 2 of the PrONTO Study. *Am J Ophthalmol.*, Vol.148, No.1, (July 2009), pp.43-58.e1, ISSN

F & Tadayoni R, (2011). Inject and extend dosing versus dosing as needed: a comparative retrospective study of ranibizumab in exudative age-related macular degeneration. *Retina*, Vol.31, No.1, (January 2011), pp.26-30, ISSN 0275-

for the treatment of choroidal neovascularization secondary to angioid streaks.

hermotherapy for myopic choroidal neovascularization: 1-year follow-up: TTT for myopic CNV. *Int Ophthalmol.,* Vol.26, No.4-5, (August-October 2005), pp.127-33,

Randomized, double-masked, sham-controlled trial of ranibizumab for neovascular age-related macular degeneration: PIER Study year 1. *Am J Ophthalmol.*, Vol.145,


Web JA. (January 2008). Genentech decision expands access to bevacizumab. In: *Ophthalmology Times,* 2011, Available from: <http://ophthalmologytimes.modernmedicine.com/ophthalmologytimes/issue/is sueDetail.jsp?id=13920>.

**9** 

*Spain* 

**Combined Therapies to Treat** 

*Fundacion Oftalmologica Mediterraneo, Valencia* 

**CNV in AMD: PDT + Anti-VEGF** 

Jorge Mataix, M. Carmen Desco, Elena Palacios and Amparo Navea

Age-related macular degeneration (AMD) causes a high incidence of morbility in the elderly. The dry forms of the disease are the most usual, but wet forms (15% of the total of cases) are responsible for 80% of AMD-related cases of severe vision loss. Age-related macular degeneration is essentially a choroidal/retinal pigment epithelium (RPE) disease which affects the overlying neurosensory retina. Formation of choroidal neovessels that penetrate the subretinal space is the main cause of vision loss. Knowing what role the vascular endothelial growth factor (VEGF) plays in angiogenesis of the formation of these neovessels is a determining factor. Ferrara's studies describe the four main biological

Since the formation of choroidal neovascularization (CNV) is a determining factor in vision loss in wet AMD, it is reasonable to expect a reduction in the risk of vision loss by inhibiting new vessel formation and preventing their growth. The efficacy of antiangiogenesis agents for this purpose has provided proof of the concept of therapy targeted at a specific molecular step in the process, namely the inhibition of VEGF (Gragouas et al. 2004). Therefore the appearance of antibodies against VEGF (anti-VEGF) brought about a considerable advance in the treatment of exudative forms of AMD. The most important effects are **regression** of existing vessels, **normalization** of surviving vessels, and **inhibition**

Antiangiogenesis agents have proved to be beneficial but are often administered late in the process when the aim of the treatment is to salvage vision rather than to prevent vision loss. One obstacle in developing a single approach to treatment stems from the possibility that AMD is the product of multiple pathologic processes. A more exciting goal is halting the process at a subclinical stage or preventing the disease in patients identified as being at risk for vision loss. Progress in isolating multiple processes responsible for disease progression is

**1. Introduction** 

of vessel growth.

functions in VEGF agents (Ferrara & Gerber, 2001):

2. Growth and proliferation of vascular endothelial cells

creating new opportunities for combination therapies.

4. Survival of immature endothelial cells by preventing apoptosis

1. Increase in vascular permeability

3. Migration of vascular endothelial cells

### **Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF**

Jorge Mataix, M. Carmen Desco, Elena Palacios and Amparo Navea *Fundacion Oftalmologica Mediterraneo, Valencia Spain* 

#### **1. Introduction**

160 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

Web JA. (January 2008). Genentech decision expands access to bevacizumab. In:

<http://ophthalmologytimes.modernmedicine.com/ophthalmologytimes/issue/is

*Ophthalmology Times,* 2011, Available from:

sueDetail.jsp?id=13920>.

Age-related macular degeneration (AMD) causes a high incidence of morbility in the elderly. The dry forms of the disease are the most usual, but wet forms (15% of the total of cases) are responsible for 80% of AMD-related cases of severe vision loss. Age-related macular degeneration is essentially a choroidal/retinal pigment epithelium (RPE) disease which affects the overlying neurosensory retina. Formation of choroidal neovessels that penetrate the subretinal space is the main cause of vision loss. Knowing what role the vascular endothelial growth factor (VEGF) plays in angiogenesis of the formation of these neovessels is a determining factor. Ferrara's studies describe the four main biological functions in VEGF agents (Ferrara & Gerber, 2001):


Since the formation of choroidal neovascularization (CNV) is a determining factor in vision loss in wet AMD, it is reasonable to expect a reduction in the risk of vision loss by inhibiting new vessel formation and preventing their growth. The efficacy of antiangiogenesis agents for this purpose has provided proof of the concept of therapy targeted at a specific molecular step in the process, namely the inhibition of VEGF (Gragouas et al. 2004). Therefore the appearance of antibodies against VEGF (anti-VEGF) brought about a considerable advance in the treatment of exudative forms of AMD. The most important effects are **regression** of existing vessels, **normalization** of surviving vessels, and **inhibition** of vessel growth.

Antiangiogenesis agents have proved to be beneficial but are often administered late in the process when the aim of the treatment is to salvage vision rather than to prevent vision loss. One obstacle in developing a single approach to treatment stems from the possibility that AMD is the product of multiple pathologic processes. A more exciting goal is halting the process at a subclinical stage or preventing the disease in patients identified as being at risk for vision loss. Progress in isolating multiple processes responsible for disease progression is creating new opportunities for combination therapies.

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 163

Fig. 2. Phases in angiogenesis in which the formation of a neovessel guided by the

which, in the long term, often plays a determining role in vision loss.

Blocking the extravascular component, like inhibiting subretinal fibrosis, can considerably reduce the morbility of the disease. Biological therapies mediated by cytokines, such as the tumor necrosis factor, ionizing radiation which does not only act on the vascular component but also on the extravascular component, corticosteroid drugs combined with anti-VEGF can improve the therapeutic response, inhibiting the development of the extracellular matrix

Currently, the most relevant therapy available is VEGF inhibition. Possibly, greater success could be achieved if other key factors in pathogenesis were also inhibited. It would be more advantageous if angiogenesis, scarring, and inflammation were targeted simultaneously. Combination approaches may not only increase overall efficacy but also reduce the potential for side effects by allowing relatively low doses to yield a greater level of efficacy than

The appearance of anti-VEGF is a revolutionary treatment in wet AMD as, for the first time, the progression of the disease can be stopped. Nevertheless, it cannot restore vision and there are still many cases that progress in spite of repeated treatment with anti-VEGF. Listed below are several limitations of anti-VEGF that make the quest for other therapies, or a

As described above, one of the functions of VEGF is favoring the survival of endothelial cells, but this function is just restricted to immature vessels in which angiogenesis

extracellular matrix is shown (Genentech image).

**3. Limitations of anti-VEGF in CNV treatment**

**3.1 Anti-VEGF agents do not affect mature vessels** 

higher doses of a single agent.

combination of therapies, necessary.

#### **2. Composition of neovessels**

Histopathologic examination of CNV shows granulation-like tissue, with the invasion of not only blood vessels, but also inflammatory and mesenchymal cells embedded in a loosely formed extracellular matrix. Although the majority of damage attributed to CNV is due to neovessel bleeding or leakage, there are other components and factors associated with CNV that influence the visual prognosis decisively.

A two-component model of CNV has been developed to offer a conceptual framework to structure combination treatments. One is the *vascular component*, which is composed of vascular endothelial cells and associated pericytes. The other is the *nonvascular component* which is made up of the remaining cells, such as the inflammatory cells, glial cells, myofibroblasts, and fibrocytes (Spaide, 2006a, 2009). Inhibiting one has the potential to inhibit, at least partially, the other due to mutual interactions between the two components and each component can potentially cause damage. Inhibiting either one would seem to offer some hope in slowing down or arresting the process, but inhibition of both would intuitively lead to the best theoretical outcome.

Blocking the vascular component is achieved mainly by administering anti-VEGF agents, although in advanced lesions with mature neovessels covered by pericytes, anti-VEGF on their own cannot make these neovessels regress. In these cases, a combination of other therapies has to be resorted to which act by means of selective mechanisms, such as blocking the platelet-derived growth factor (PDGF) to target the pericytes, or a non-selective attack mechanism, such as ionizing radiation which is explained further on (Jain RK, 2005) in this chapter.

Fig. 1. CNV components: vascular and nonvascular components

Histopathologic examination of CNV shows granulation-like tissue, with the invasion of not only blood vessels, but also inflammatory and mesenchymal cells embedded in a loosely formed extracellular matrix. Although the majority of damage attributed to CNV is due to neovessel bleeding or leakage, there are other components and factors associated with CNV

A two-component model of CNV has been developed to offer a conceptual framework to structure combination treatments. One is the *vascular component*, which is composed of vascular endothelial cells and associated pericytes. The other is the *nonvascular component* which is made up of the remaining cells, such as the inflammatory cells, glial cells, myofibroblasts, and fibrocytes (Spaide, 2006a, 2009). Inhibiting one has the potential to inhibit, at least partially, the other due to mutual interactions between the two components and each component can potentially cause damage. Inhibiting either one would seem to offer some hope in slowing down or arresting the process, but inhibition of both would

Blocking the vascular component is achieved mainly by administering anti-VEGF agents, although in advanced lesions with mature neovessels covered by pericytes, anti-VEGF on their own cannot make these neovessels regress. In these cases, a combination of other therapies has to be resorted to which act by means of selective mechanisms, such as blocking the platelet-derived growth factor (PDGF) to target the pericytes, or a non-selective attack mechanism, such as ionizing radiation which is explained further on (Jain RK, 2005) in this

**2. Composition of neovessels** 

that influence the visual prognosis decisively.

intuitively lead to the best theoretical outcome.

Fig. 1. CNV components: vascular and nonvascular components

chapter.

Fig. 2. Phases in angiogenesis in which the formation of a neovessel guided by the extracellular matrix is shown (Genentech image).

Blocking the extravascular component, like inhibiting subretinal fibrosis, can considerably reduce the morbility of the disease. Biological therapies mediated by cytokines, such as the tumor necrosis factor, ionizing radiation which does not only act on the vascular component but also on the extravascular component, corticosteroid drugs combined with anti-VEGF can improve the therapeutic response, inhibiting the development of the extracellular matrix which, in the long term, often plays a determining role in vision loss.

Currently, the most relevant therapy available is VEGF inhibition. Possibly, greater success could be achieved if other key factors in pathogenesis were also inhibited. It would be more advantageous if angiogenesis, scarring, and inflammation were targeted simultaneously. Combination approaches may not only increase overall efficacy but also reduce the potential for side effects by allowing relatively low doses to yield a greater level of efficacy than higher doses of a single agent.

#### **3. Limitations of anti-VEGF in CNV treatment**

The appearance of anti-VEGF is a revolutionary treatment in wet AMD as, for the first time, the progression of the disease can be stopped. Nevertheless, it cannot restore vision and there are still many cases that progress in spite of repeated treatment with anti-VEGF. Listed below are several limitations of anti-VEGF that make the quest for other therapies, or a combination of therapies, necessary.

#### **3.1 Anti-VEGF agents do not affect mature vessels**

As described above, one of the functions of VEGF is favoring the survival of endothelial cells, but this function is just restricted to immature vessels in which angiogenesis

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 165

In this way, the angiogenic factors basically act as survival factors, while antiangiogenic factors act as apoptosis-inducing factors in the context of endothelial expansion, which is an immature endothelium. Thus the balance between the inducing and inhibiting factors determines the destiny of immature vessels, but they do not bear an influence on mature vessels that are in a quiescent state due to the interaction between endothelial cells, pericytes, and the extracellular matrix (Jimenez Cuenca B, 2003). Therefore, if the neovessels are already mature, they will not react to VEGF, nor will they respond to anti-VEGF (Benjamin et. al., 1999). This fact underpins the value of combined therapies like photodynamic therapy (PDT) with verteporfin, which is necessary to destroy the architecture of different mature components of the neovascular

Despite their beneficial clinical effects in AMD, anti-VEGF therapies are ineffective in regressing existing lesions. Endothelial cells and pericytes that form the structure of new vascular tissue typically do not regress with VEGF inhibition alone. This limitation is confirmed by data from the key prospective, randomized clinical trials with Ranibizumab, such as PIER, ANCHOR, and MARINA (Regillo et al., 2008; Brown et al., 2006; Rosenfeld et al., 2006) which did not produce any significant evidence of neovascular regression despite improvement in visual acuity. There are no changes from baseline in the CNV area. This lack of regression is also consistent with experimental models in which monotherapy with anti-VEGF agents inhibits new vascular formation but has little effect on existing capillaries. Despite the importance of VEGF agents in the cascade of events that stimulates and sustains new vascular formation, VEGF inhibition may have limited effects on existing neovascular tissue once subsequent molecular events are triggered, making inhibition of additional

Recent publications refer to the possible existence of tachyphylaxis after use of intravitreal Bevacizumab. Forooghian and co-workers describe a decrease in the duration of the beneficial effect, and even a lack of response, after a mean of eight intravitreal injections (Forooghian et al., 2009) in six (n = 59) patients with AMD treated with Bevacizumab in

Currently, the existence of tachyphylaxis is under discussion and its mechanism is unknown. This author poses the possibility of an autoimmune mechanism after a patient, suffering from uveitis, presented tachyphylaxis immediately after intravitreal injection. It must be remembered that Bevacizumab, in spite of being a "humanized" antibody for decreasing immunogenicity, could trigger the formation of new anti-Bevacizumab

In addition, it should be considered that always inhibiting the same vascular pathway may potentiate other pathogenic pathways, such as the inflammatory pathway, or that it may increase other cytokines involved in wet AMD. Certainly, a greater inflammatory activity and a proliferation of macrophages in the membranes that were surgically extracted with prior Bevacizumab treatment were observed compared with those extracted without prior

membrane that do not respond to anti-VEGF.

**3.3 Tachyphylaxis** 

monotherapy.

intravitreal treatment (Tatar et al., 2009).

**3.2 Anti-VEGF agents do not decrease the CNV size** 

molecular steps essential to build on the benefits of anti-VEGF therapies.

antibodies after repeated treatments; this hypothesis has yet to be studied.

necessarily requires the activation of survival pathways to maintain the condition of the vessels (Gerber et al., 1998). Nonetheless, the mechanisms involved in the maturing process of the vessels, such as pericyte coverage and the formation of interaction between endothelial and periendotherlial cells with the basal membrane, free the endothelial cell from the requirement of the survival function of VEGF.

Fig. 3. Composition of a mature blood vessel, made up of endothelial cells, pericyte coverage, and the interactions between them.

Fig. 4. When the factors that inhibit angiogenesis predominate over those that induce it, two things can occur, depending on the context. If the vessel is mature, it will show no response as it is quiescent and is not affected by the effect of the antiangiogenic drugs. However, if the vessels are immature, with no pericyte coverage, the angiogenesis inhibitors will favor apoptosis and the regression of the vessel. When proangiogenic factors predominate the immature vessel survives and stabilizes.

In this way, the angiogenic factors basically act as survival factors, while antiangiogenic factors act as apoptosis-inducing factors in the context of endothelial expansion, which is an immature endothelium. Thus the balance between the inducing and inhibiting factors determines the destiny of immature vessels, but they do not bear an influence on mature vessels that are in a quiescent state due to the interaction between endothelial cells, pericytes, and the extracellular matrix (Jimenez Cuenca B, 2003). Therefore, if the neovessels are already mature, they will not react to VEGF, nor will they respond to anti-VEGF (Benjamin et. al., 1999). This fact underpins the value of combined therapies like photodynamic therapy (PDT) with verteporfin, which is necessary to destroy the architecture of different mature components of the neovascular membrane that do not respond to anti-VEGF.

#### **3.2 Anti-VEGF agents do not decrease the CNV size**

Despite their beneficial clinical effects in AMD, anti-VEGF therapies are ineffective in regressing existing lesions. Endothelial cells and pericytes that form the structure of new vascular tissue typically do not regress with VEGF inhibition alone. This limitation is confirmed by data from the key prospective, randomized clinical trials with Ranibizumab, such as PIER, ANCHOR, and MARINA (Regillo et al., 2008; Brown et al., 2006; Rosenfeld et al., 2006) which did not produce any significant evidence of neovascular regression despite improvement in visual acuity. There are no changes from baseline in the CNV area. This lack of regression is also consistent with experimental models in which monotherapy with anti-VEGF agents inhibits new vascular formation but has little effect on existing capillaries.

Despite the importance of VEGF agents in the cascade of events that stimulates and sustains new vascular formation, VEGF inhibition may have limited effects on existing neovascular tissue once subsequent molecular events are triggered, making inhibition of additional molecular steps essential to build on the benefits of anti-VEGF therapies.

#### **3.3 Tachyphylaxis**

164 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

necessarily requires the activation of survival pathways to maintain the condition of the vessels (Gerber et al., 1998). Nonetheless, the mechanisms involved in the maturing process of the vessels, such as pericyte coverage and the formation of interaction between endothelial and periendotherlial cells with the basal membrane, free the endothelial cell

Fig. 3. Composition of a mature blood vessel, made up of endothelial cells, pericyte

Fig. 4. When the factors that inhibit angiogenesis predominate over those that induce it, two things can occur, depending on the context. If the vessel is mature, it will show no response as it is quiescent and is not affected by the effect of the antiangiogenic drugs. However, if the vessels are immature, with no pericyte coverage, the angiogenesis inhibitors will favor apoptosis and the regression of the vessel. When proangiogenic factors predominate the

from the requirement of the survival function of VEGF.

coverage, and the interactions between them.

immature vessel survives and stabilizes.

Recent publications refer to the possible existence of tachyphylaxis after use of intravitreal Bevacizumab. Forooghian and co-workers describe a decrease in the duration of the beneficial effect, and even a lack of response, after a mean of eight intravitreal injections (Forooghian et al., 2009) in six (n = 59) patients with AMD treated with Bevacizumab in monotherapy.

Currently, the existence of tachyphylaxis is under discussion and its mechanism is unknown. This author poses the possibility of an autoimmune mechanism after a patient, suffering from uveitis, presented tachyphylaxis immediately after intravitreal injection. It must be remembered that Bevacizumab, in spite of being a "humanized" antibody for decreasing immunogenicity, could trigger the formation of new anti-Bevacizumab antibodies after repeated treatments; this hypothesis has yet to be studied.

In addition, it should be considered that always inhibiting the same vascular pathway may potentiate other pathogenic pathways, such as the inflammatory pathway, or that it may increase other cytokines involved in wet AMD. Certainly, a greater inflammatory activity and a proliferation of macrophages in the membranes that were surgically extracted with prior Bevacizumab treatment were observed compared with those extracted without prior intravitreal treatment (Tatar et al., 2009).

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 167

In a study in which 82 patients, treated with Ranibizumab monotherapy, were monitored in a follow-up of two to six years, reported that the rate of fibrosis was 50% and the rate of atrophy of the retinal pigment epithelium was 40%; 8% percent had both. The fibrosis may have been caused by ongoing inflammation and by maturation of vascular tissue. Hemorrhage above or below the subretinal pigment epithelium may have played a contributing role. Fibrosis was often observed after several years of antiangiogenesis

These findings reinforce other evidence that fibrosis may be an important additional target to expand or preserve the benefits of Ranibizumab and other VEGF inhibitors in the treatment of AMD. Several strategies are being pursued. These include antagonists of integrin, inhibitors of the mammalian target of rapamycin, vascular disrupting agents, and radiation. Control of atrophy, which is generally observed at an earlier stage of AMD progression than fibrosis, is another potential target for improving outcome. The candidate targets for preventing atrophy include neurotrophic factors, free radical scavengers, and retinol binding competitors.

Even the maximum inhibition of VEGF does not stop vascular growth due to the presence of redundant signaling pathways. Controlling one pathway, the inhibition of neovascularization is relatively modest due to the presence of redundant signaling pathways. The combination of molecules inhibit different parts of the angiogenic process and provide more profound inhibition of neovascularization relative to blocking a single

When one of two inhibitors of angiogenesis was used in experimental studies (Dorrell et al., 2007) complete inhibition of new vessel formation was achieved in a small proportion of animals. In contrast, complete inhibition of neovascularization was observed in more than

Transscleral and intravitreal injections are alternative methods of local delivery. These methods may reduce the risk of systemic absorption, because topical therapy results in a significant amount of the drug draining away from the eye through the nasal lacrimal duct into the gut. Transscleral and intravitreal injections may also increase the percentage of the dose that reaches a posterior target. The efficacy of this approach is well documented with antiangiogenic therapies for AMD, but it is not risk-free for the patient, and moreover, it requires surgical administration, which in many cases can cause saturation of operating rooms and delays to the detriment of the patient. Moreover, as it is a chronic disease, retreatment is often necessary which increases these problems. Association of anti-VEGF with other therapies can be useful in reducing the number of anti-VEGF doses without

Not least important is the potential for unwanted effects on the biologic function controlled by drug targets, such as prolonged suppression of a complex molecule like VEGF, which while being a key factor in causing CNV associated with AMD, also plays an important role

therapy even among responders (Kaiser PK, 2009c).

**3.5 Several pathogenic pathways involved** 

proangiogenic signal (Frielander, 2009).

reducing its effectiveness.

as a neuroprotectant in the mature retina.

half of the animals treated with triple combination therapy.

**3.6 Route of administration, number and frequency of doses** 

Complement inhibition may be another viable strategy (Heier JS, 2009).

Furthermore a better response to intravitreal corticoids has been observed in patients who stop responding to anti-VEGF intravitreal injections (Schaal et al., 2008). Therefore we pose a future problem regarding the use of anti-VEGF, i.e., how to avoid this occurrence. One of the most appropriate options would be to find the way to reduce the number of intravitreal injections in the treatment of AMD and, to date, the only way is with combined therapies.

#### **3.4 Anti-VEGF agents do not act on fibrosis and atrophy**

Long-term follow-up of patients who participated in the initial studies of anti-VEGF therapies suggests that late visual loss is often caused by processes that seem to be independent of neovascularization, particularly fibrosis and atrophy. Although more effective antiangiogenesis treatments, including combination strategies, for better blockade of new vessel formation are likely to improve outcome, it is appropriate to expand targets to other pathophysiologic processes associated with AMD. It is necessary to incorporate therapies that block fibrosis and inhibit atrophy or other pathophysiologic processes not directly related to neovascularization.

In Ranibizumab trials (Rosenfeld et al., 2006; Brown et al., 2006), protection against visual loss was highly significant relative to controls during a follow-up of twelve and 24 months. Gains in visual acuity were much smaller: only 34% achieved >15 letter gain at twelve months or 24 months on the most effective dose of Ranibizumab and 65% of patients with very modest gains, no gains, or visual loss over the course of these studies. The subgroup of patients with a loss of three or more lines of visual acuity tended to have better visual acuity than average at baseline, but a larger area of CNV and a larger area of CNV leakage. Over the course of treatment, these patients had a greater growth in total lesion area and more retinal pigment epithelium abnormalities.

Fig. 5. (A) Retinograph and OCT of subretinal fibrosis of an AMD patient treated with anti-VEGF. (B) Autofluorescence of macular atrophy in the context of wet AMD that had already been treated. In both cases, the evolution of visual acuity was poor despite inactivating the lesion with repeated doses of anti-VEGF.

Furthermore a better response to intravitreal corticoids has been observed in patients who stop responding to anti-VEGF intravitreal injections (Schaal et al., 2008). Therefore we pose a future problem regarding the use of anti-VEGF, i.e., how to avoid this occurrence. One of the most appropriate options would be to find the way to reduce the number of intravitreal injections in the treatment of AMD and, to date, the only way is with combined therapies.

Long-term follow-up of patients who participated in the initial studies of anti-VEGF therapies suggests that late visual loss is often caused by processes that seem to be independent of neovascularization, particularly fibrosis and atrophy. Although more effective antiangiogenesis treatments, including combination strategies, for better blockade of new vessel formation are likely to improve outcome, it is appropriate to expand targets to other pathophysiologic processes associated with AMD. It is necessary to incorporate therapies that block fibrosis and inhibit atrophy or other pathophysiologic processes not

In Ranibizumab trials (Rosenfeld et al., 2006; Brown et al., 2006), protection against visual loss was highly significant relative to controls during a follow-up of twelve and 24 months. Gains in visual acuity were much smaller: only 34% achieved >15 letter gain at twelve months or 24 months on the most effective dose of Ranibizumab and 65% of patients with very modest gains, no gains, or visual loss over the course of these studies. The subgroup of patients with a loss of three or more lines of visual acuity tended to have better visual acuity than average at baseline, but a larger area of CNV and a larger area of CNV leakage. Over the course of treatment, these patients had a greater growth in total lesion area and more

Fig. 5. (A) Retinograph and OCT of subretinal fibrosis of an AMD patient treated with anti-VEGF. (B) Autofluorescence of macular atrophy in the context of wet AMD that had already been treated. In both cases, the evolution of visual acuity was poor despite inactivating the

**3.4 Anti-VEGF agents do not act on fibrosis and atrophy** 

directly related to neovascularization.

retinal pigment epithelium abnormalities.

**A B** 

lesion with repeated doses of anti-VEGF.

In a study in which 82 patients, treated with Ranibizumab monotherapy, were monitored in a follow-up of two to six years, reported that the rate of fibrosis was 50% and the rate of atrophy of the retinal pigment epithelium was 40%; 8% percent had both. The fibrosis may have been caused by ongoing inflammation and by maturation of vascular tissue. Hemorrhage above or below the subretinal pigment epithelium may have played a contributing role. Fibrosis was often observed after several years of antiangiogenesis therapy even among responders (Kaiser PK, 2009c).

These findings reinforce other evidence that fibrosis may be an important additional target to expand or preserve the benefits of Ranibizumab and other VEGF inhibitors in the treatment of AMD. Several strategies are being pursued. These include antagonists of integrin, inhibitors of the mammalian target of rapamycin, vascular disrupting agents, and radiation. Control of atrophy, which is generally observed at an earlier stage of AMD progression than fibrosis, is another potential target for improving outcome. The candidate targets for preventing atrophy include neurotrophic factors, free radical scavengers, and retinol binding competitors. Complement inhibition may be another viable strategy (Heier JS, 2009).

#### **3.5 Several pathogenic pathways involved**

Even the maximum inhibition of VEGF does not stop vascular growth due to the presence of redundant signaling pathways. Controlling one pathway, the inhibition of neovascularization is relatively modest due to the presence of redundant signaling pathways. The combination of molecules inhibit different parts of the angiogenic process and provide more profound inhibition of neovascularization relative to blocking a single proangiogenic signal (Frielander, 2009).

When one of two inhibitors of angiogenesis was used in experimental studies (Dorrell et al., 2007) complete inhibition of new vessel formation was achieved in a small proportion of animals. In contrast, complete inhibition of neovascularization was observed in more than half of the animals treated with triple combination therapy.

#### **3.6 Route of administration, number and frequency of doses**

Transscleral and intravitreal injections are alternative methods of local delivery. These methods may reduce the risk of systemic absorption, because topical therapy results in a significant amount of the drug draining away from the eye through the nasal lacrimal duct into the gut. Transscleral and intravitreal injections may also increase the percentage of the dose that reaches a posterior target. The efficacy of this approach is well documented with antiangiogenic therapies for AMD, but it is not risk-free for the patient, and moreover, it requires surgical administration, which in many cases can cause saturation of operating rooms and delays to the detriment of the patient. Moreover, as it is a chronic disease, retreatment is often necessary which increases these problems. Association of anti-VEGF with other therapies can be useful in reducing the number of anti-VEGF doses without reducing its effectiveness.

Not least important is the potential for unwanted effects on the biologic function controlled by drug targets, such as prolonged suppression of a complex molecule like VEGF, which while being a key factor in causing CNV associated with AMD, also plays an important role as a neuroprotectant in the mature retina.

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 169

appear to be the primary site of angiogenic stimulation (Schmidt-Erfurth et al., 2003). This suggests that combining PDT with anti-VEGF for decreasing the PDT response is advisable. The increase in the formation of VEGF, VEGF-3, and PEDF can favor the growth of CNV after initial PDT treatment. This has been observed constantly in our series of 262 cases treated with PDT with a 48-month follow-up. We noted an increase in the CNV size throughout the observation period. This growth was nearly 60% of the total size increase at

Photodynamic therapy is very effective in the initial control of CNV because it achieves almost 100% closure of the neovessels in all patients in a period of seven days to one month (Miller et al., 1999). Its side effects include hypoxia, stimulation of inflammatory factors, and upregulation of VEGF expression (Schmith-Erfurth et al., 2001), which can be prevented by associating an anti-VEGF. Although monotherapy with PDT achieves inactivation of the lesion, it does not inhibit subsequent growth, bringing about a loss of vision that in many

The benefits of PDT are documented in a great variety of cases with CNV due to AMD and there is encouraging evidence of improved outcomes when this angioocclusive modality is combined with antiangiogenic agents (Schmidt-Erfurth et al., 2009). It is known that treatment with verteporfin produces hypoperfusion in the treated area and that concomitant use of anti-VEGF can prolong this effect. Moreover, numerous analyses show minimal evidence that there is association with visual deterioration or other adverse effects. Furthermore, hypoperfusion helps to reduce recanalization of CNV and permits neuronal recovery by decreasing exposure to oxygen and oxidative radicals. The reduced need for frequent retreatments clearly has a major appeal due to the lower costs associated with fewer interventions and reduced burden of clinical monitoring and diagnostic reevaluations

Various clinical assays have been performed with different designs in which combined treatment has been compared with monotherapy. A previous study - PROTECT (Schmidt-Erfurth, 2008) – evaluated the safety and efficacy of administering PDT and Ranibizumab on the same day. Photodynamic therapy was applied and an hour later the intravitreal injection was given. Photodynamic therapy was repeated every three months in accordance with the investigator's opinion and Ranibizumab was administered the first three months, then as required. The study served to show that combined treatment performed on the same day is

The FOCUS study (Heier, 2006) was designed to evaluate, in wet AMD with predominantly classic CNV the safety and efficacy of the combination of Ranibizumab and PDT as the first treatment, followed by monthly Ranibizumab for the first twelve months and PDT every three months according to the investigator's opinion. The control group only received PDT and a simulation injection. After twelve months, the study group showed 90.5% of eyes had lost less than 15 letters as opposed to 67.9% in the control group. The combined treatment group received a mean of 1.32 PDT and the control group 4 PDT per year the first year. After 24 months there was a difference of 12.4 letters in favor of the combined treatment

month three after the first PDT treatment (Mataix et al. 2009).

cases is difficult to recover (Awan, et al., 2009).

**4.2 Clinical trials combining PDT + Ranibizumab** 

(Schmidt-Erfurth et al., 2009).

safe and effective.

group.

Exudative AMD is a sub-acute process. Its natural progress from the first symptoms of CNV to scar formation takes over a year in most cases, but can even be active for years (Holz et al., 2004). In fact, the disease evolves to final subfoveal scarring, including the cases where the disease was extrafoveal initially. A well-known fact is that unfortunately, sometimes after years of thermal laser treatment of an extrafoveal lesion, there is foveal recurrence. Antiangiogenic drugs can prevent the growth of new blood vessels but it is not known how long antiangiogenic activity must be kept up to prevent CNV reactivation; it may be needed for years.

There is good justification for considering combination strategies in AMD to build on the initial success achieved with VEGF inhibitors, but combination strategies impose considerable challenges. The frequency of intravitreal injections causes significant difficulties in terms of clinical management and patient convenience and available devices for implantation do not seem to be viable for chronic treatment in their current form.

#### **4. Combined therapies with anti-VEGF**

Mediation synergies are used in medicine to potentiate the effect that two or more drugs provide separately, acting on the disease from a different etiopathogenic approach. As AMD is a complex process, it seems logical to focus its treatment from different physiopathological strategies. The combination of agents with different action mechanisms can give rise to a synergic effect, a lower number of overall treatments, and a greater duration of the response when compared with Ranibizumab monotherapy, while the outcome on visual acuity persists.

#### **4.1 Action mechanisms: PDT and anti-VEGF**

Photodynamic therapy with verteporfin has been used for years in the treatment of CNV in AMD and its action mechanism has been described repeatedly. Briefly, the action of verteporfin with non-thermal laser in the macular area where the CNV is present triggers processes that lead to apoptosis (Granville et al., 2001), alters the lipids of the cellular membranes of the endothelium, triggers plaquetary aggregation and thrombosis, and increases vascular permeability, blood stasis, and tissue hypoxia (Fingar, 1996). There is an increase in VEGF expression in this process which is the cause of the growth and reactivation of the common membrane before the third month; association of an anti-VEGF inhibits this effect.

Pharmacologic inhibition of VEGF-A decreases the proliferation of endothelial cells and recruitment of others, such as leukocytes, which can express the cytokines and proteases necessary to develop and maintain neovessels (Witmer, 2003; Ferrara, 2003). However, once neovascularization is stabilized, it will not respond to anti-VEGF treatment (Benjamin, 1999). This would explain the added benefit of associating PDT to destroy the architecture of the different components of the neovascular membrane that do not respond to anti-VEGF.

In 2003, Schmidt-Erfurth evaluated the impact PDT has on the expression and distribution of VEGF, VEGF receptor (VEGFR)-3, and pigment epithelium-derived factor (PEDF) after applying it to the retina. Said author reported that PDT using verteporfin induces a reproducible angiogenic response in elderly human eyes. Vascular endothelial growth factor, VEGFR-3, and PEDF expression is enhanced after PDT. Choroidal endothelial cells

Exudative AMD is a sub-acute process. Its natural progress from the first symptoms of CNV to scar formation takes over a year in most cases, but can even be active for years (Holz et al., 2004). In fact, the disease evolves to final subfoveal scarring, including the cases where the disease was extrafoveal initially. A well-known fact is that unfortunately, sometimes after years of thermal laser treatment of an extrafoveal lesion, there is foveal recurrence. Antiangiogenic drugs can prevent the growth of new blood vessels but it is not known how long antiangiogenic activity must be kept up to prevent CNV reactivation; it may be needed

There is good justification for considering combination strategies in AMD to build on the initial success achieved with VEGF inhibitors, but combination strategies impose considerable challenges. The frequency of intravitreal injections causes significant difficulties in terms of clinical management and patient convenience and available devices

Mediation synergies are used in medicine to potentiate the effect that two or more drugs provide separately, acting on the disease from a different etiopathogenic approach. As AMD is a complex process, it seems logical to focus its treatment from different physiopathological strategies. The combination of agents with different action mechanisms can give rise to a synergic effect, a lower number of overall treatments, and a greater duration of the response when compared with Ranibizumab monotherapy, while the

Photodynamic therapy with verteporfin has been used for years in the treatment of CNV in AMD and its action mechanism has been described repeatedly. Briefly, the action of verteporfin with non-thermal laser in the macular area where the CNV is present triggers processes that lead to apoptosis (Granville et al., 2001), alters the lipids of the cellular membranes of the endothelium, triggers plaquetary aggregation and thrombosis, and increases vascular permeability, blood stasis, and tissue hypoxia (Fingar, 1996). There is an increase in VEGF expression in this process which is the cause of the growth and reactivation of the common membrane before the third month; association of an anti-VEGF

Pharmacologic inhibition of VEGF-A decreases the proliferation of endothelial cells and recruitment of others, such as leukocytes, which can express the cytokines and proteases necessary to develop and maintain neovessels (Witmer, 2003; Ferrara, 2003). However, once neovascularization is stabilized, it will not respond to anti-VEGF treatment (Benjamin, 1999). This would explain the added benefit of associating PDT to destroy the architecture of the different components of the neovascular membrane that do not respond to anti-VEGF.

In 2003, Schmidt-Erfurth evaluated the impact PDT has on the expression and distribution of VEGF, VEGF receptor (VEGFR)-3, and pigment epithelium-derived factor (PEDF) after applying it to the retina. Said author reported that PDT using verteporfin induces a reproducible angiogenic response in elderly human eyes. Vascular endothelial growth factor, VEGFR-3, and PEDF expression is enhanced after PDT. Choroidal endothelial cells

for implantation do not seem to be viable for chronic treatment in their current form.

**4. Combined therapies with anti-VEGF** 

**4.1 Action mechanisms: PDT and anti-VEGF** 

outcome on visual acuity persists.

inhibits this effect.

for years.

appear to be the primary site of angiogenic stimulation (Schmidt-Erfurth et al., 2003). This suggests that combining PDT with anti-VEGF for decreasing the PDT response is advisable. The increase in the formation of VEGF, VEGF-3, and PEDF can favor the growth of CNV after initial PDT treatment. This has been observed constantly in our series of 262 cases treated with PDT with a 48-month follow-up. We noted an increase in the CNV size throughout the observation period. This growth was nearly 60% of the total size increase at month three after the first PDT treatment (Mataix et al. 2009).

Photodynamic therapy is very effective in the initial control of CNV because it achieves almost 100% closure of the neovessels in all patients in a period of seven days to one month (Miller et al., 1999). Its side effects include hypoxia, stimulation of inflammatory factors, and upregulation of VEGF expression (Schmith-Erfurth et al., 2001), which can be prevented by associating an anti-VEGF. Although monotherapy with PDT achieves inactivation of the lesion, it does not inhibit subsequent growth, bringing about a loss of vision that in many cases is difficult to recover (Awan, et al., 2009).

The benefits of PDT are documented in a great variety of cases with CNV due to AMD and there is encouraging evidence of improved outcomes when this angioocclusive modality is combined with antiangiogenic agents (Schmidt-Erfurth et al., 2009). It is known that treatment with verteporfin produces hypoperfusion in the treated area and that concomitant use of anti-VEGF can prolong this effect. Moreover, numerous analyses show minimal evidence that there is association with visual deterioration or other adverse effects. Furthermore, hypoperfusion helps to reduce recanalization of CNV and permits neuronal recovery by decreasing exposure to oxygen and oxidative radicals. The reduced need for frequent retreatments clearly has a major appeal due to the lower costs associated with fewer interventions and reduced burden of clinical monitoring and diagnostic reevaluations (Schmidt-Erfurth et al., 2009).

#### **4.2 Clinical trials combining PDT + Ranibizumab**

Various clinical assays have been performed with different designs in which combined treatment has been compared with monotherapy. A previous study - PROTECT (Schmidt-Erfurth, 2008) – evaluated the safety and efficacy of administering PDT and Ranibizumab on the same day. Photodynamic therapy was applied and an hour later the intravitreal injection was given. Photodynamic therapy was repeated every three months in accordance with the investigator's opinion and Ranibizumab was administered the first three months, then as required. The study served to show that combined treatment performed on the same day is safe and effective.

The FOCUS study (Heier, 2006) was designed to evaluate, in wet AMD with predominantly classic CNV the safety and efficacy of the combination of Ranibizumab and PDT as the first treatment, followed by monthly Ranibizumab for the first twelve months and PDT every three months according to the investigator's opinion. The control group only received PDT and a simulation injection. After twelve months, the study group showed 90.5% of eyes had lost less than 15 letters as opposed to 67.9% in the control group. The combined treatment group received a mean of 1.32 PDT and the control group 4 PDT per year the first year. After 24 months there was a difference of 12.4 letters in favor of the combined treatment group.

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 171

Bevacizumab intravitreal injections was used. The mean improvement obtained was 1.8 lines in almost ten months with a low number of retreatments. A vitrectomy was associated to inject a greater volume of liquid. In a record of cases published recently, 1,073 patients were treated with PDT and Bevacizumab as required, with 1.6 PDT and three injections in twelve months, achieving 82% of patients with a loss of fewer than three lines (Kaiser,

An increase in the treatment-free interval was observed in the PDT + anti-VEGF combination. Wan (Wan et al., 2010) in their study on 174 patients with AMD treated with PDT followed by intravitreal injection of Bevacizumab, obtained a mean of 193 days of treatment-free interval, and 52% of the patients did not require postinduction retreatment in the ten months of follow-up. Moreover, other studies report stabilizing the lesion with one single dose of PDT induction + anti-VEGF after twelve months follow-up. The percentage of cases varies in different studies ranging from 39.6% to 46% to 48% (Mataix, 2010; Navea

A systematic review published recently (Das et al., 2011) establishes that intravitreal treatment with anti-VEGF obtains an increase in vision in AMD patients. Combination with PDT brings about a reduction in the number and frequency of retreatments and maintains the improvement in the long term. It seems fairly conclusive that combined treatment for neovascular AMD is a therapeutic option for diseases which do not respond to monotherapy. Moreover, it has the advantage of minimizing the risk monotherapy does have, that of potentiating other chronification pathways of the neovascular disease as it could allow compensatory stimulation of other pathogenic mechanisms of the disease. Tao and Jonas used a combination of Bevacizumab and high-dose-triamcinolone-acetonide in a group of 29 patients who were being treated with Bevacizumab in monotherapy and obtaining no visual or anatomic response. They achieved a visual improvement and reduction in macular thickness (Tao & Jonas, 2010). However, Rudinsky reported finding no benefit in combination therapy in a retrospective study which compared 139 eyes treated with Bevacizumab with 236 treated with PDT + Bevacizumab. The monotherapy eyes showed an improvement of 5.05 letters versus 4.8 letters with combination therapy; there was no difference between the groups. The monotherapy eyes received 3.32 injections

versus 3.14 injections in the combination therapy group (Rudinsky et al., 2010).

number of retreatments when compared with anti-VEGF monotherapy.

A recent study (Forte et al., 2011) compared PDT + dexamethasone + anti-VEGF (Ranibizumab or Bevacizumab) triple therapy with Ranibizumab or Bevacizumab monotherapy. Sixty-one eyes were included in the first group and 40 in the second. The mean follow-up was between 14 and 16 months. The triple-therapy group required fewer treatments (1.92 vs 3.12); furthermore, on average, this group took longer to require the first retreatment (5.4 vs 3.6 months). There was a significant improvement in vision and foveal thickness in both groups, therefore it can be concluded that triple therapy reduces the

Use of reduced-fluence PDT in combination with anti-VEGF is another method that is obtaining good outcomes. Spielberg treated 27 cases with reduced-fluence PDT followed by intravitreal Ranibizumab on the same day. Retreatments administered with Ranibizumab during the 24-month follow-up stabilized 84% of the patients' vision or improved it at month 24 (Spielberg & Leys, 2010). A prospective comparative study was performed on 85 AMD patients divided into two groups, one treated with intravitreal Bevacizumab (IVB)

2009a).

2009; Smith, 2008).

The most interesting assays are the SUMMIT with its two groups, the DENALI which was carried out in the United States and Canada, and the MONTBLANC which was performed in Europe. They were designed to determine whether PDT combined with Ranibizumab was better than monotherapy with Ranibizumab and they included patients with all types of lesions. They were divided randomly into two groups in the MONTBLANC study: in one, PDT was performed and basal intravitreal Ranibizumab and two more injections of Ranibizumab were administered; subsequent treatments were as required and PDT was associated in accordance with the investigator's opinion every three months. Monotherapy and simulating PDT were used in the control group. In the American group, a third group with combined treatment of low fluence PDT was added.

According to the results after twelve months of the MONTBLANC study presented at the European Retina Society in Amsterdam in 2010, the differences between combined and monotherapy treatment were slight in overall terms. The visual behavior was similar between the study and control groups. Neither was there a very significant difference regarding the need for retreatments in the two groups, although with combination therapy a tendency towards a decrease in repeated treatments with Ranibizumab was observed. After twelve months, the mean change in best-corrected visual acuity (BCVA) was +2.5 in the combined treatment group and +4.4 in the monotherapy group. Over 50% of the patients in the two groups gained at least one line of vision compared with their basal value. There was a mean improvement in VA of +2 letters in the combined group and +1.6 letters in the monotherapy group after twelve months in the predominantly classic lesions. Patients with ≤2 area of disc (AD) lesions experienced a mean improvement in VA of +9.7 letters in the combination group and +7.1 letters in the monotherapy group after twelve months.

The patients in the combination group received, on average, 0.3 times fewer Ranibizumab injections. The mean number of treatment repetitions with Ranibizumab after the loading phase in the combination group was 1.8 compared with 2.2 in the Ranibizumab monotherapy group. A tendency towards a decrease in repeated treatments with Lucentis was observed in the combination group. Patients with predominantly classic lesions and smaller lesions who received combined treatment seemed to present better visual results with combined treatment than with monotherapy. It was also observed that the monotherapy group conserved the vision obtained after the initial three loading injections when these were followed by individualized therapy and, on average, fewer injections were necessary.

In the RADICAL (*Reduced Fluence Visudyne-Anti-VEGF-Dexamethasone In Combination for AMD Lesions)* study, other combinations, as opposed to monotherapy, were analyzed which included PDT with reduced fluence + Ranibizumab, PDT with reduced fluence + Ranibizumab + dexamethasone, and PDT with very reduced fluence + Ranibizumab + dexamethasone. In general, there was a tendency to fewer repetitions in the combined groups, with similar outcomes and adverse effects in all the groups (Hughes et al., 2009).

#### **4.3 Other studies**

Several studies have been published in which combination treatments were used. Most of them were made up of small groups or with short follow-ups. An interesting study by Augustin's group (2007) included 104 eyes; a triple treatment of PDT, dexamethasone, and

The most interesting assays are the SUMMIT with its two groups, the DENALI which was carried out in the United States and Canada, and the MONTBLANC which was performed in Europe. They were designed to determine whether PDT combined with Ranibizumab was better than monotherapy with Ranibizumab and they included patients with all types of lesions. They were divided randomly into two groups in the MONTBLANC study: in one, PDT was performed and basal intravitreal Ranibizumab and two more injections of Ranibizumab were administered; subsequent treatments were as required and PDT was associated in accordance with the investigator's opinion every three months. Monotherapy and simulating PDT were used in the control group. In the American group, a third group

According to the results after twelve months of the MONTBLANC study presented at the European Retina Society in Amsterdam in 2010, the differences between combined and monotherapy treatment were slight in overall terms. The visual behavior was similar between the study and control groups. Neither was there a very significant difference regarding the need for retreatments in the two groups, although with combination therapy a tendency towards a decrease in repeated treatments with Ranibizumab was observed. After twelve months, the mean change in best-corrected visual acuity (BCVA) was +2.5 in the combined treatment group and +4.4 in the monotherapy group. Over 50% of the patients in the two groups gained at least one line of vision compared with their basal value. There was a mean improvement in VA of +2 letters in the combined group and +1.6 letters in the monotherapy group after twelve months in the predominantly classic lesions. Patients with ≤2 area of disc (AD) lesions experienced a mean improvement in VA of +9.7 letters in the

combination group and +7.1 letters in the monotherapy group after twelve months.

The patients in the combination group received, on average, 0.3 times fewer Ranibizumab injections. The mean number of treatment repetitions with Ranibizumab after the loading phase in the combination group was 1.8 compared with 2.2 in the Ranibizumab monotherapy group. A tendency towards a decrease in repeated treatments with Lucentis was observed in the combination group. Patients with predominantly classic lesions and smaller lesions who received combined treatment seemed to present better visual results with combined treatment than with monotherapy. It was also observed that the monotherapy group conserved the vision obtained after the initial three loading injections when these were followed by individualized therapy and, on average, fewer injections were

In the RADICAL (*Reduced Fluence Visudyne-Anti-VEGF-Dexamethasone In Combination for AMD Lesions)* study, other combinations, as opposed to monotherapy, were analyzed which included PDT with reduced fluence + Ranibizumab, PDT with reduced fluence + Ranibizumab + dexamethasone, and PDT with very reduced fluence + Ranibizumab + dexamethasone. In general, there was a tendency to fewer repetitions in the combined groups, with similar outcomes and adverse effects in all the groups (Hughes et al., 2009).

Several studies have been published in which combination treatments were used. Most of them were made up of small groups or with short follow-ups. An interesting study by Augustin's group (2007) included 104 eyes; a triple treatment of PDT, dexamethasone, and

with combined treatment of low fluence PDT was added.

necessary.

**4.3 Other studies** 

Bevacizumab intravitreal injections was used. The mean improvement obtained was 1.8 lines in almost ten months with a low number of retreatments. A vitrectomy was associated to inject a greater volume of liquid. In a record of cases published recently, 1,073 patients were treated with PDT and Bevacizumab as required, with 1.6 PDT and three injections in twelve months, achieving 82% of patients with a loss of fewer than three lines (Kaiser, 2009a).

An increase in the treatment-free interval was observed in the PDT + anti-VEGF combination. Wan (Wan et al., 2010) in their study on 174 patients with AMD treated with PDT followed by intravitreal injection of Bevacizumab, obtained a mean of 193 days of treatment-free interval, and 52% of the patients did not require postinduction retreatment in the ten months of follow-up. Moreover, other studies report stabilizing the lesion with one single dose of PDT induction + anti-VEGF after twelve months follow-up. The percentage of cases varies in different studies ranging from 39.6% to 46% to 48% (Mataix, 2010; Navea 2009; Smith, 2008).

A systematic review published recently (Das et al., 2011) establishes that intravitreal treatment with anti-VEGF obtains an increase in vision in AMD patients. Combination with PDT brings about a reduction in the number and frequency of retreatments and maintains the improvement in the long term. It seems fairly conclusive that combined treatment for neovascular AMD is a therapeutic option for diseases which do not respond to monotherapy. Moreover, it has the advantage of minimizing the risk monotherapy does have, that of potentiating other chronification pathways of the neovascular disease as it could allow compensatory stimulation of other pathogenic mechanisms of the disease. Tao and Jonas used a combination of Bevacizumab and high-dose-triamcinolone-acetonide in a group of 29 patients who were being treated with Bevacizumab in monotherapy and obtaining no visual or anatomic response. They achieved a visual improvement and reduction in macular thickness (Tao & Jonas, 2010). However, Rudinsky reported finding no benefit in combination therapy in a retrospective study which compared 139 eyes treated with Bevacizumab with 236 treated with PDT + Bevacizumab. The monotherapy eyes showed an improvement of 5.05 letters versus 4.8 letters with combination therapy; there was no difference between the groups. The monotherapy eyes received 3.32 injections versus 3.14 injections in the combination therapy group (Rudinsky et al., 2010).

A recent study (Forte et al., 2011) compared PDT + dexamethasone + anti-VEGF (Ranibizumab or Bevacizumab) triple therapy with Ranibizumab or Bevacizumab monotherapy. Sixty-one eyes were included in the first group and 40 in the second. The mean follow-up was between 14 and 16 months. The triple-therapy group required fewer treatments (1.92 vs 3.12); furthermore, on average, this group took longer to require the first retreatment (5.4 vs 3.6 months). There was a significant improvement in vision and foveal thickness in both groups, therefore it can be concluded that triple therapy reduces the number of retreatments when compared with anti-VEGF monotherapy.

Use of reduced-fluence PDT in combination with anti-VEGF is another method that is obtaining good outcomes. Spielberg treated 27 cases with reduced-fluence PDT followed by intravitreal Ranibizumab on the same day. Retreatments administered with Ranibizumab during the 24-month follow-up stabilized 84% of the patients' vision or improved it at month 24 (Spielberg & Leys, 2010). A prospective comparative study was performed on 85 AMD patients divided into two groups, one treated with intravitreal Bevacizumab (IVB)

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 173

**letters** 

**Gain ≥ 25 letters** 

mg. (monthly) 94,6 % 33,8 % +7,2 13 Ranibizumab

mg. (monthly) 96,4% 40,3% +11,3 13 Ranibizumab

87% 18% +2,5

88% 26% +5

92,3% 32,7% +7,2

82% 36% +6

16% (24 months) +7,2 (24 months)

Bevacizumab +4,8 3,14

84% (24 months)

96,9% 1 PDT

90,5% 23,8% +4,9 1,4 PDT

95,2% 19% +5,7 1,46 PDT

**VA change from baseline** 

**Number of treatments (average)** 

4 Ranibizumab

13 Ranibizumab

1,7 PDT 4,8

Ranibizumab

2 Bevacizumab

1,22 PDT 2,37

1,6 PDT 3,2

Ranibizumab

Bevacizumab

Bevacizumab

Bevacizumab (12 months)

Bevacizumab (24 months)

5,1

7,1

**(months) Treatment Loss < 15** 

PDT + Ranibizumab 0,5mg monthly

PDT + Ranibizumab 0,5mg monthly

PDT +

PDT + Bevacizumab 1,25mg (single initial doses) retreatments as required

PDT + Ranibizumab (single initial doses) retreatments as required

PDT + Ranibizumab (single initial doses) retreatments as required

PDT (1/2 fluence) + Ranibizumab 0,5mg (3 initial doses) retreatments as required

Ranibizumab (3 initial doses) retreatments as required

PDT (1/2 fluence) + Ranibizumab 0,5mg (3 initial doses) retreatments as required

**<sup>N</sup>Follow-up** 

**MARINA** 240 12 Ranibizumab 0,5

**ANCHOR** 146 12 Ranibizumab 0,5

**PROTECT** 32 4

**FOCUS** 106 12

**BLANC** 122 12

**RADICAL** 43 12

**Navea et. al.** 63 12

**Mataix et. al.** 53 12

**Kaiser et. al.** 701 12

**al.** 27 12 / 24

**al.** 236 12 PDT +

Table 1. Summary of studies with PDT + Anti-VEGF

**Rudinsky et.** 

**Spielberg et.** 

**MONT** 

monotherapy and the other with IVB combined with low-fluence PDT (300 mW/cm2 for 83 s, 25 J/cm2 ) with a twelve-month follow-up. The combination of IVB with low fluence PDT for the treatment of classic or predominantly classic neovascular AMD worked in a synergistic fashion with a significant reduction in IVB reinjection rate (Costagliola et al., 2009).

Kovacs recently published a retrospective analysis of triple combination therapy with IVB, posterior sub-tenon's triamcinolon acetonide and low fluence verteporfin PDT with good visual results and a reduction in macular thickness with a twelve-month follow-up (Kovacs et al., 2011).

#### **4.4 Our experience**

Our group has considerable experience in treatment combining PDT with anti-VEGF. In 2006 we began to treat patients with wet AMD using this method. A sample of this work appears in two publications showing the results of two groups using different treatments, one PDT + Bevacizumab and the other PDT + Ranibizumab.

The study groups included patients with active subfoveal and juxtafoveal CNV secondary to AMD, naïve cases, initial BCVA ≥ 20/400, and maximum lesion size under 5.400 µm defined on fluorescein angiography (FA).

The treatment included a single, initial dose of PDT + Bevacizumab/Ranibizumab. Criteria for retreatment were based on OCT, BCVA, and FA; an increase in central retinal thickness of over 100 μm or the presence of subretinal fluid was a criterion for retreatment. Loss of more than five letters of vision since the previous visit or the presence of new macular bleeding was also a criterion for retreatment if any kind of fluid was present on the OCT. In both situations an FA was performed and treatment with PDT + Bevacizumab/Ranibizumab was provided if a CNV increase or fluorescein leakage was observed. If the FA did not show a CNV increase or fluorescein leakage, treatment was provided with Ranibizumab alone. Photodynamic treatment was only provided when over three months had elapsed since the previous PDT. If development of macular atrophic changes seemed to be the cause of vision loss, it was not treated.

We studied 53 eyes of 53 patients treated with PDT + Ranibizumab and 63 eyes of 63 patients treated with PDT + Bevacizumab, with a twelve-month follow-up. The demographic characteristics and the characteristics of the CNV were similar in both groups. The CNV localization was mainly subfoveal in both groups, with a mean size of 2386 and 2064 μm in the Ranibizumab and Bevacizumab groups, respectively.

**Evolution of retinal thickness:** The OCT baseline central retinal thickness was 372 μm. It decreased to 251 μm in the first month of treatment and remained the same throughout the follow-up, reaching a mean thickness of 254 μm twelve months later in the group with the Ranibizumab combination, with a mean reduction of -118 μm. The OCT baseline central retinal thickness was 357 μm, decreasing to 246 μm in the first month of treatment and reaching a mean thickness of 227 μm twelve months later in the group with the Bevacizumab combination, with a mean reduction of -129 μm. Separate analysis of the two groups with Student's t-test showed the reduction in retinal thickness was statistically significant (p<0.05) from the first month, remaining the same throughout the year.

monotherapy and the other with IVB combined with low-fluence PDT (300 mW/cm2 for 83 s, 25 J/cm2 ) with a twelve-month follow-up. The combination of IVB with low fluence PDT for the treatment of classic or predominantly classic neovascular AMD worked in a synergistic fashion with a significant reduction in IVB reinjection rate (Costagliola et al.,

Kovacs recently published a retrospective analysis of triple combination therapy with IVB, posterior sub-tenon's triamcinolon acetonide and low fluence verteporfin PDT with good visual results and a reduction in macular thickness with a twelve-month follow-up (Kovacs

Our group has considerable experience in treatment combining PDT with anti-VEGF. In 2006 we began to treat patients with wet AMD using this method. A sample of this work appears in two publications showing the results of two groups using different treatments,

The study groups included patients with active subfoveal and juxtafoveal CNV secondary to AMD, naïve cases, initial BCVA ≥ 20/400, and maximum lesion size under 5.400 µm defined

The treatment included a single, initial dose of PDT + Bevacizumab/Ranibizumab. Criteria for retreatment were based on OCT, BCVA, and FA; an increase in central retinal thickness of over 100 μm or the presence of subretinal fluid was a criterion for retreatment. Loss of more than five letters of vision since the previous visit or the presence of new macular bleeding was also a criterion for retreatment if any kind of fluid was present on the OCT. In both situations an FA was performed and treatment with PDT + Bevacizumab/Ranibizumab was provided if a CNV increase or fluorescein leakage was observed. If the FA did not show a CNV increase or fluorescein leakage, treatment was provided with Ranibizumab alone. Photodynamic treatment was only provided when over three months had elapsed since the previous PDT. If development of macular atrophic

We studied 53 eyes of 53 patients treated with PDT + Ranibizumab and 63 eyes of 63 patients treated with PDT + Bevacizumab, with a twelve-month follow-up. The demographic characteristics and the characteristics of the CNV were similar in both groups. The CNV localization was mainly subfoveal in both groups, with a mean size of 2386 and

**Evolution of retinal thickness:** The OCT baseline central retinal thickness was 372 μm. It decreased to 251 μm in the first month of treatment and remained the same throughout the follow-up, reaching a mean thickness of 254 μm twelve months later in the group with the Ranibizumab combination, with a mean reduction of -118 μm. The OCT baseline central retinal thickness was 357 μm, decreasing to 246 μm in the first month of treatment and reaching a mean thickness of 227 μm twelve months later in the group with the Bevacizumab combination, with a mean reduction of -129 μm. Separate analysis of the two groups with Student's t-test showed the reduction in retinal thickness was statistically

significant (p<0.05) from the first month, remaining the same throughout the year.

one PDT + Bevacizumab and the other PDT + Ranibizumab.

changes seemed to be the cause of vision loss, it was not treated.

2064 μm in the Ranibizumab and Bevacizumab groups, respectively.

2009).

et al., 2011).

**4.4 Our experience** 

on fluorescein angiography (FA).


Table 1. Summary of studies with PDT + Anti-VEGF

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 175

Fig. 7. Percentage of visual acuity variations throughout the follow-up in patients treated with PDT + Ranibizumab: 92.3% of cases lost fewer than 15 letters after twelve months.

Fig. 8. Evolution of OCT from the beginning of the treatment until the end of the follow-up

twelve months later.

Visual loss was avoided in 78.8% of cases and 57.7% gained vision.

**Visual Acuity Evolution:** In the Ranibizumab group, the mean initial BCVA was 8.26 lines which increased to 10.3 lines in the first month of treatment. This gain was maintained until the sixth month, after which it decreased slightly to 9.72 lines twelve month later, thus obtaining a mean increase of 1.21 lines, which is equivalent to a gain of 6.05 letters after twelve months. This slight decrease is due to the severe and occasional loss of vision in a few cases after the sixth month which affected the total mean. The mean initial BCVA was 8.41 lines in the Bevacizumab group and increased from the first month of treatment to 9.38 lines. This gain was maintained throughout the twelve months, undergoing small variations and reaching 9.54 lines, obtaining a mean increase of 1.12 lines after twelve months, which is a gain of 5.6 letters. The percentage of cases that lost 15 letters was 95.2% in the PDT + Bevacizumab group and 92.3% in the PDT + Ranibizumab group. Visual gain was 58.7% and 57.7%, respectively (Figures 6, 7).

Fig. 6. Percentage of visual acuity variations throughout the follow-up in patients treated with PDT + Bevacizumab: 95.2% of cases lost fewer than 15 letters after twelve months. Visual loss was avoided in 79.3% of cases and 58.7% gained vision.

The distribution of the lines of vision between the beginning and end of the follow-up is statistically significant (p-value <0.001) in both groups. The Mann-Whitney test concludes that the visual gain is significantly better (p-value <0.05) in the first six months in the Ranibizumab group, but there were no differences between the two groups after one year.

*Retreatments:* A record of the number of treatments was kept throughout the study for both groups. The patients in the group treated with Bevacizumab received a mean of 1.46 therapies and 1.92 intravitreal injections, and those treated with Ranibizumab received a mean of 1.23 therapies and 2.38 intravitreal injections. The Mann-Whitney test showed that the Bevacizumab group received significantly more therapies (p-value <0.05), but there was no difference in the number of intravitreal injections. In 21 cases (39.6%) only a single initial combination therapy was required in the Ranibizumab group versus 29 cases (46%) in the Bevacizumab group to keep the lesion stable until the end of the follow-up. In the Ranibizumab group, 77.4% of the patients were treated with a maximum of three injections and 79.2% of the patients needed a single PDT treatment at the initiation of the treatment. In the Bevacizumab group, 87.3% and 61.9% of the patients were treated, respectively.

**Visual Acuity Evolution:** In the Ranibizumab group, the mean initial BCVA was 8.26 lines which increased to 10.3 lines in the first month of treatment. This gain was maintained until the sixth month, after which it decreased slightly to 9.72 lines twelve month later, thus obtaining a mean increase of 1.21 lines, which is equivalent to a gain of 6.05 letters after twelve months. This slight decrease is due to the severe and occasional loss of vision in a few cases after the sixth month which affected the total mean. The mean initial BCVA was 8.41 lines in the Bevacizumab group and increased from the first month of treatment to 9.38 lines. This gain was maintained throughout the twelve months, undergoing small variations and reaching 9.54 lines, obtaining a mean increase of 1.12 lines after twelve months, which is a gain of 5.6 letters. The percentage of cases that lost 15 letters was 95.2% in the PDT + Bevacizumab group and 92.3% in the PDT + Ranibizumab group. Visual gain was 58.7% and

Fig. 6. Percentage of visual acuity variations throughout the follow-up in patients treated with PDT + Bevacizumab: 95.2% of cases lost fewer than 15 letters after twelve months.

The distribution of the lines of vision between the beginning and end of the follow-up is statistically significant (p-value <0.001) in both groups. The Mann-Whitney test concludes that the visual gain is significantly better (p-value <0.05) in the first six months in the Ranibizumab group, but there were no differences between the two groups after one year. *Retreatments:* A record of the number of treatments was kept throughout the study for both groups. The patients in the group treated with Bevacizumab received a mean of 1.46 therapies and 1.92 intravitreal injections, and those treated with Ranibizumab received a mean of 1.23 therapies and 2.38 intravitreal injections. The Mann-Whitney test showed that the Bevacizumab group received significantly more therapies (p-value <0.05), but there was no difference in the number of intravitreal injections. In 21 cases (39.6%) only a single initial combination therapy was required in the Ranibizumab group versus 29 cases (46%) in the Bevacizumab group to keep the lesion stable until the end of the follow-up. In the Ranibizumab group, 77.4% of the patients were treated with a maximum of three injections and 79.2% of the patients needed a single PDT treatment at the initiation of the treatment. In

the Bevacizumab group, 87.3% and 61.9% of the patients were treated, respectively.

Visual loss was avoided in 79.3% of cases and 58.7% gained vision.

57.7%, respectively (Figures 6, 7).

Fig. 7. Percentage of visual acuity variations throughout the follow-up in patients treated with PDT + Ranibizumab: 92.3% of cases lost fewer than 15 letters after twelve months. Visual loss was avoided in 78.8% of cases and 57.7% gained vision.

Fig. 8. Evolution of OCT from the beginning of the treatment until the end of the follow-up twelve months later.

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 177

signals to neovascular endothelial cells and hence makes them resistant to VEGF (Bergers et al., 2003). Pericytes are essential in vascular maturation so their inhibition is important in inhibiting neovascularization and the regression of new mature vessels. **Anti-PDGF (Platelet-derived growth factor)** treatment strips away pericytes to leave the endothelial cells unprotected and vulnerable to anti-VEGF treatment (Erber R. et al., 2004). The combination of anti-VEGF and anti–PDGF produces inhibition and regression of corneal and choroidal neovascularization compared with anti-VEGF treatment alone. In models of pathologic tumor angiogenesis, strategies involving both anti-VEGF and anti-PDGF have

also produced regression when an anti-VEGF therapy alone failed (Jo N et al., 2006).

Ranibizumab effect (Boyer DS et al., 2009).

**myofibroblasts, and fibrocytes)** 

**Platelet-derived growth factor**, which has an important role in recruiting the pericytes critical to maturation of vessel walls, may also be a viable target to augment the effects of a VEGF inhibitor. A recent phase 1 clinical trial with anti-PDGF (E10030) included patients with subfoveal CNV who received three monthly doses of E10030 in combination with a standard dose of Ranibizumab. The preliminary findings reveal a reduction in neovascular size (neovascular regression) in all patients. This regression is associated with a marked improvement in visual acuity, gain ≥ 15 ETDRS letters: 4 weeks (32%) 12 weeks (59%) and gain in numbers of letters: 4 weeks (10.9%) 12 weeks (14%). However, it is not yet clear whether the improvement was due to the E10030/Ranibizumab combination or simply a

The inhibition of **Insulin-like growth factor** could be another option in the treatment of neovessels. It leads to endothelial cell proliferation and inhibits apoptosis of endothelial cells, the nicotinic acetylcholine receptor, which also induces endothelial cell migration, and

Antiangiogenesis agents are effective for preventing progression of CNV in a substantial proportion of patients, although regression is not typically observed. Experimental studies indicate that newly formed capillaries are no longer susceptible to regression with anti-VEGF agents within about two weeks after formation. Antiangiogenesis agents may still be effective for preventing the development of additional capillaries or reducing leakage in vessels invading the retina, but the persistence of CNV may stimulate inflammation or other pathologic processes that eventually result in vision loss due to the formation of fibrosis.

**Radiation therapy** has long been used to control fibrosis in a variety of tissues. In AMD, radiation may be particularly attractive because there is evidence of synergistic inhibition of neovascularization when radiation is combined with antiangiogenesis drugs (Nieder C. et al., 2007). Historically, radiation monotherapy sufficient to eradicate CNV effectively has been associated with a modest benefit for AMD**.** The growing evidence that antiangiogenic agents can increase the antitumor efficacy of radiotherapy includes studies in animal models: the combination of radiation and antiangiogenesis agents had a greater effect in reducing tumor regrowth than either alone (Gorski DH et al., 1999). In another animal study, the use of anti-VEGF and anti-PDGF agents in combination with radiation showed a significantly greater antitumor effect relative to radiation alone (Timke C et al., 2008). Mammalian target of rapamycin (mTOR) inhibitors (Sirolimus) radiosensitize cancer cells *in* 

tubular binding proteins, which govern endothelial cell shape formation.

Prevention of fibrosis is essential to the preservation of VA.

**5.2 The nonvascular component (inflammatory cells, cytokines, glial cells,** 

Our results suggest that a combination of PDT and anti-VEGF is a good option for treating CNV in AMD more effectively by maintaining good visual results and decreasing the need for retreatments.

#### **5. Other therapies**

The etiology of AMD is multifactorial and there are several mechanisms that can lead to irreversible loss of vision. To achieve a complete therapy for CNV, we should not simply focus on neovessels, but rather act on both the vascular component (mature and immature vessels) and the non-vascular component (inflammatory cells, cytokines, glial cells, myofibroblasts, fibrocytes, etc.).

#### **5.1 The vascular component (immature and mature vessels)**

The antiangiogenesis agents currently used in the treatment of AMD inhibit VEGF by blocking the growth factor in the extra-cellular space, thereby preventing access to its receptor. Several VEGF inhibitors, including **Bevacizumab and Ranibizumab**, have demonstrated excellent safety and efficacy in exudative AMD (Rosenfeld PJ et al., 2006; Spaide RF et al., 2006b). The current strategy of blocking VEGF in the extracellular space may be an inadequate approach for long-term control of AMD. Combining drugs that act at different points of the angiogenesis pathway has the potential to build on the benefits of extracellular VEGF inhibitors, but a more profound inhibition of the disease process may require activity in additional pathways of the disease.

**VEGF Trap-Eye** is a fully human soluble VEGF receptor fusion protein that binds all forms of VEGF-A along with the related placental growth factor (PlGF). VEGF Trap-Eye is made by fusing two different domains from VEGF receptors 1 and 2 onto a human Fc fragment. VEGF Trap-Eye has tighter VEGF binding than the natural receptor and has greater affinity than the current VEGF inhibitors (Steward & Rosenfeld, 2007). Two parallel Phase 3 trials have been developed in patients with wet AMD (VIEW 1 and VIEW 2). VEGF Trap-Eye is being dosed at 0.5 mg every four weeks, 2 mg every four weeks, and 2 mg every eight weeks in direct comparison with Ranibizumab administered at 0.5 mg every four weeks during the first year of the studies. The primary endpoint was statistical non-inferiority in the proportion of patients who maintained (or improved) vision over 52 weeks compared to Ranibizumab. A generally favorable safety profile was observed for both VEGF Trap-Eye and Ranibizumab.

**Small interfering RNA agents,** such as **RTP-801i and Bevasiranib**, which turn off target genes, are extremely promising in a variety of therapeutic areas. **Bevasiranib** is designed to block the production of VEGF directly by inhibiting the messenger RNA from the VEGF gene. Studies in mice have demonstrated that Bevasiranib can inhibit and regress ocular neovascularization (Reich et al., 2003). Studies in rabbits have shown that an intravitreal injection of Bevasiranib achieved good distribution in the retina and in RPE (Dejneka NS et al., 2008). The agent is well distributed after intravitreal injection and well tolerated by human subjects (Karagiannis & El-Osta, 2005). A Phase 3 Clinical Trial tests this agent in combination with Ranibizumab because potentially the drug may prevent further production of VEGF while Ranibizumab blocks the VEGF that is present.

Experimental models show that monotherapy with anti-VEGF agents inhibits new vascular formation but has little effect on existing capillaries. Pericyte coverage provides survival

Our results suggest that a combination of PDT and anti-VEGF is a good option for treating CNV in AMD more effectively by maintaining good visual results and decreasing the need

The etiology of AMD is multifactorial and there are several mechanisms that can lead to irreversible loss of vision. To achieve a complete therapy for CNV, we should not simply focus on neovessels, but rather act on both the vascular component (mature and immature vessels) and the non-vascular component (inflammatory cells, cytokines, glial cells,

The antiangiogenesis agents currently used in the treatment of AMD inhibit VEGF by blocking the growth factor in the extra-cellular space, thereby preventing access to its receptor. Several VEGF inhibitors, including **Bevacizumab and Ranibizumab**, have demonstrated excellent safety and efficacy in exudative AMD (Rosenfeld PJ et al., 2006; Spaide RF et al., 2006b). The current strategy of blocking VEGF in the extracellular space may be an inadequate approach for long-term control of AMD. Combining drugs that act at different points of the angiogenesis pathway has the potential to build on the benefits of extracellular VEGF inhibitors, but a more profound inhibition of the disease process may

**VEGF Trap-Eye** is a fully human soluble VEGF receptor fusion protein that binds all forms of VEGF-A along with the related placental growth factor (PlGF). VEGF Trap-Eye is made by fusing two different domains from VEGF receptors 1 and 2 onto a human Fc fragment. VEGF Trap-Eye has tighter VEGF binding than the natural receptor and has greater affinity than the current VEGF inhibitors (Steward & Rosenfeld, 2007). Two parallel Phase 3 trials have been developed in patients with wet AMD (VIEW 1 and VIEW 2). VEGF Trap-Eye is being dosed at 0.5 mg every four weeks, 2 mg every four weeks, and 2 mg every eight weeks in direct comparison with Ranibizumab administered at 0.5 mg every four weeks during the first year of the studies. The primary endpoint was statistical non-inferiority in the proportion of patients who maintained (or improved) vision over 52 weeks compared to Ranibizumab. A generally favorable safety profile was observed for both VEGF Trap-Eye and Ranibizumab.

**Small interfering RNA agents,** such as **RTP-801i and Bevasiranib**, which turn off target genes, are extremely promising in a variety of therapeutic areas. **Bevasiranib** is designed to block the production of VEGF directly by inhibiting the messenger RNA from the VEGF gene. Studies in mice have demonstrated that Bevasiranib can inhibit and regress ocular neovascularization (Reich et al., 2003). Studies in rabbits have shown that an intravitreal injection of Bevasiranib achieved good distribution in the retina and in RPE (Dejneka NS et al., 2008). The agent is well distributed after intravitreal injection and well tolerated by human subjects (Karagiannis & El-Osta, 2005). A Phase 3 Clinical Trial tests this agent in combination with Ranibizumab because potentially the drug may prevent further

Experimental models show that monotherapy with anti-VEGF agents inhibits new vascular formation but has little effect on existing capillaries. Pericyte coverage provides survival

production of VEGF while Ranibizumab blocks the VEGF that is present.

for retreatments.

**5. Other therapies**

myofibroblasts, fibrocytes, etc.).

**5.1 The vascular component (immature and mature vessels)** 

require activity in additional pathways of the disease.

signals to neovascular endothelial cells and hence makes them resistant to VEGF (Bergers et al., 2003). Pericytes are essential in vascular maturation so their inhibition is important in inhibiting neovascularization and the regression of new mature vessels. **Anti-PDGF (Platelet-derived growth factor)** treatment strips away pericytes to leave the endothelial cells unprotected and vulnerable to anti-VEGF treatment (Erber R. et al., 2004). The combination of anti-VEGF and anti–PDGF produces inhibition and regression of corneal and choroidal neovascularization compared with anti-VEGF treatment alone. In models of pathologic tumor angiogenesis, strategies involving both anti-VEGF and anti-PDGF have also produced regression when an anti-VEGF therapy alone failed (Jo N et al., 2006).

**Platelet-derived growth factor**, which has an important role in recruiting the pericytes critical to maturation of vessel walls, may also be a viable target to augment the effects of a VEGF inhibitor. A recent phase 1 clinical trial with anti-PDGF (E10030) included patients with subfoveal CNV who received three monthly doses of E10030 in combination with a standard dose of Ranibizumab. The preliminary findings reveal a reduction in neovascular size (neovascular regression) in all patients. This regression is associated with a marked improvement in visual acuity, gain ≥ 15 ETDRS letters: 4 weeks (32%) 12 weeks (59%) and gain in numbers of letters: 4 weeks (10.9%) 12 weeks (14%). However, it is not yet clear whether the improvement was due to the E10030/Ranibizumab combination or simply a Ranibizumab effect (Boyer DS et al., 2009).

The inhibition of **Insulin-like growth factor** could be another option in the treatment of neovessels. It leads to endothelial cell proliferation and inhibits apoptosis of endothelial cells, the nicotinic acetylcholine receptor, which also induces endothelial cell migration, and tubular binding proteins, which govern endothelial cell shape formation.

#### **5.2 The nonvascular component (inflammatory cells, cytokines, glial cells, myofibroblasts, and fibrocytes)**

Antiangiogenesis agents are effective for preventing progression of CNV in a substantial proportion of patients, although regression is not typically observed. Experimental studies indicate that newly formed capillaries are no longer susceptible to regression with anti-VEGF agents within about two weeks after formation. Antiangiogenesis agents may still be effective for preventing the development of additional capillaries or reducing leakage in vessels invading the retina, but the persistence of CNV may stimulate inflammation or other pathologic processes that eventually result in vision loss due to the formation of fibrosis. Prevention of fibrosis is essential to the preservation of VA.

**Radiation therapy** has long been used to control fibrosis in a variety of tissues. In AMD, radiation may be particularly attractive because there is evidence of synergistic inhibition of neovascularization when radiation is combined with antiangiogenesis drugs (Nieder C. et al., 2007). Historically, radiation monotherapy sufficient to eradicate CNV effectively has been associated with a modest benefit for AMD**.** The growing evidence that antiangiogenic agents can increase the antitumor efficacy of radiotherapy includes studies in animal models: the combination of radiation and antiangiogenesis agents had a greater effect in reducing tumor regrowth than either alone (Gorski DH et al., 1999). In another animal study, the use of anti-VEGF and anti-PDGF agents in combination with radiation showed a significantly greater antitumor effect relative to radiation alone (Timke C et al., 2008). Mammalian target of rapamycin (mTOR) inhibitors (Sirolimus) radiosensitize cancer cells *in* 

Combined Therapies to Treat CNV in AMD: PDT + Anti-VEGF 179

implicated in the formation of drusen in primate models and human postmortem specimens (Anderson DH et al., 2002), patients with membranoproliferative glomerulonephritis type II, a disorder characterized by uncontrolled complement cascade activation, develop drusen histologically identical to drusen associated with AMD (Mullins RF et al., 2001). These findings support the potential for adding therapies directed at complement activation to those that already demonstrate activity in AMD, but it is not yet clear what incites the complement cascade or how to inhibit it at its source. Therefore, to attack the complement pathway, it may be necessary to address both membrane attack complex and C5a, while preserving the beneficial antimicrobial function of C3 (Giese MJ et al., 1994). There are numerous questions about where and when to block complement to inhibit best progression of AMD. An experimental treatment, known as ARC 1905, has been associated with the inhibition of C5a and C5b-9. In experimental models, this **inhibitor of C5aR** has demonstrated measurable activity in reducing the influx of neutrophils and macrophages and has also been associated

**Mammalian target of rapamycin,** a protein kinase linked to a variety of gene transcriptions and protein production, including VEGF, is strongly implicated in a number of proliferative processes and is a targetable mediator of fibrosis. Palomid 529 (mTOR inhibitor) has demonstrated a strong antifibrotic effect in retinal fibrosis models, including laser-induced retinopathy. The antifibrotic activity of this inhibitor has been measured, across a variety of endpoints, including inhibition of the inflammatory response as well as the extent of the fibrotic scar. Other mammalian targets of rapamycin inhibitors, such as sirolimus, have demonstrated good antiangiogenic, antiinflammatory, and antifibrotic effects (Chiang GC et al., 2007) and inhibit the response to interleukin-2 (IL-2) and thereby block activation of T-

The potential for a single therapy to control the complex process of AMD seems to be relatively remote. It is not clear that a combination of different agents, working on the same pathway of extracellular VEGF inhibition, will reduce AMD progression, but there may be a strong potential for additive or synergistic effects from combining drugs with independent mechanisms. The current strategy of blocking VEGF in the extracellular space may be an inadequate approach for long-term control of AMD. Combining drugs that act at different points of the angiogenesis pathway has the potential to build on the benefits of extracellular VEGF inhibitors, but a more profound inhibition of the disease process may require activity

Adamis AP. (2009). The Rationale for drug combinations in Age related macular

Anderson DH.; Mullins RF.; Hageman GS. &J ohnson LV. (2002). A role for local

degeneration. *Retina*, Vol. 29, No. Supplement 6, (June 2009), pp. 42-44, ISSN 0275-

inflammation in the formation of drusen in the aging eye. *American Journal Ophthalmology*, Vol. 134, No. 3, (September 2002), pp.411–431, ISSN 0002-9394

with suppression of CNV (Adamis AP, 2009).

and B-cells.

**6. Conclusion** 

**7. References** 

004X

in additional pathways of the disease.

*vitro* and several studies have demonstrated their ability to radiosensitize *in vivo* (Shinohara ET et al., 2005). In a Phase I recent report, CNV was irradiated in a few patients with strontium 90 delivered by a specialized, 20-gauge, intravitreal probe that is placed over the lesion intraoperatively**.** All patients underwent a vitrectomy before radiotherapy and were treated with Bevacizumab before and after radiation. In a 4-minute exposure, the estimated dose at the lesion was 24 Gy**.** Results were excellent, with 76% of patients gaining at least 10 letters of visual acuity. Most of the responders required no more than one additional injection of Bevacizumab over the first year of follow-up.

**Integrins** as a mediator of adhesion both between cells and between cells and extracellular matrix, have a role in a variety of proliferative processes, including fibrosis. Integrin also seems to have a direct influence on proliferative kinase signalingare, a mediator of adhesion between cells and extracellular matrix. It is a transmembrane protein that binds to extracellular matrix proteins (fibronectin) allowing cell adhesion and cytoskeletal organization. The α5β1 is especially important in pathologic angiogenesis (not in normal vasculature) (Kim S et al., 2000). Many of the cellular effects of VEGF are duplicated downstream in the angiogenic cascade by the interaction between the transmembrane integrin α5β1receptor and its natural ligand, fibronectin. In addition to VEGF, the interaction of integrin α5β1 with fibronectin is critical to endothelial cell survival. Integrin α5β1 has been shown to be upregulated in all the cells associated with AMD pathogenesis, including endothelial cells, retinal pigment epithelium cells, macrophages, and fibroblasts. This implicates this molecule in multiple pathogenic processes involved in AMD, including neovascularization, vascular leakage, and inflammation (Klatt K et al., 2007). Two studies have demonstrated that once neovascular tissue begins to grow, the extracellular matrix needs to adhere to the neovascular endothelial cells for them to survive (Hynes RO, 2002; Hodivala-Dilke et al., 2003). Inhibiting the ligation of integrin α5β1 and fibronectin may disrupt the process of neovascularization, regardless of the upstream growth factor pathway. **Volociximab** (M200), a chimeric monoclonal antibody targeting integrin α5β1 to block its ligation of fibronectin, has robustly inhibited human umbilical vein endothelial cell tube formation in laboratory tests. It does so regardless of an initial growth factor stimulant. It has also inhibited neovascularization in primate choroid tissue and tumor angiogenesis in rabbits (Ramakrishnan V et al., 2006; Bhaskar V et al., 2008).

**Vascular disrupting agents** also have potential for the inhibition of fibrosis as well as formation of new blood vessels. Unlike antiangiogenesis agents that block formation of new blood vessels, vascular-disrupting agents attack newly formed endothelium by disrupting connectivity between cells. This activity is expected to be complementary to anti-VEGF agents because it takes place at a later stage of neovascularization. It may also exert an important antifibrotic effect. A vascular-disrupting agent called **combretastatin** A4P (CA4P) has been evaluated in a Phase 1 study in humans with myopic macular degeneration, where it demonstrated relatively modest effects, but the characteristics of AMD may be more suitable for its activity.

Compared with age-matched controls, individuals with AMD demonstrate elevations in a variety of systemic biomarkers of inflammation, including activated monocytes and interleukin-6 (Vine AK et al., 2005 & Seddon et al., 2005). An increased risk of AMD in individuals with polymorphisms in their genes coding for the complement regulatory proteins is another signal that complement driven inflammation is perhaps an important mediator of this disease (Klein RJ et al., 2005). Although terminal elements of the complement pathway are implicated in the formation of drusen in primate models and human postmortem specimens (Anderson DH et al., 2002), patients with membranoproliferative glomerulonephritis type II, a disorder characterized by uncontrolled complement cascade activation, develop drusen histologically identical to drusen associated with AMD (Mullins RF et al., 2001). These findings support the potential for adding therapies directed at complement activation to those that already demonstrate activity in AMD, but it is not yet clear what incites the complement cascade or how to inhibit it at its source. Therefore, to attack the complement pathway, it may be necessary to address both membrane attack complex and C5a, while preserving the beneficial antimicrobial function of C3 (Giese MJ et al., 1994). There are numerous questions about where and when to block complement to inhibit best progression of AMD. An experimental treatment, known as ARC 1905, has been associated with the inhibition of C5a and C5b-9. In experimental models, this **inhibitor of C5aR** has demonstrated measurable activity in reducing the influx of neutrophils and macrophages and has also been associated with suppression of CNV (Adamis AP, 2009).

**Mammalian target of rapamycin,** a protein kinase linked to a variety of gene transcriptions and protein production, including VEGF, is strongly implicated in a number of proliferative processes and is a targetable mediator of fibrosis. Palomid 529 (mTOR inhibitor) has demonstrated a strong antifibrotic effect in retinal fibrosis models, including laser-induced retinopathy. The antifibrotic activity of this inhibitor has been measured, across a variety of endpoints, including inhibition of the inflammatory response as well as the extent of the fibrotic scar. Other mammalian targets of rapamycin inhibitors, such as sirolimus, have demonstrated good antiangiogenic, antiinflammatory, and antifibrotic effects (Chiang GC et al., 2007) and inhibit the response to interleukin-2 (IL-2) and thereby block activation of Tand B-cells.

#### **6. Conclusion**

178 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

*vitro* and several studies have demonstrated their ability to radiosensitize *in vivo* (Shinohara ET et al., 2005). In a Phase I recent report, CNV was irradiated in a few patients with strontium 90 delivered by a specialized, 20-gauge, intravitreal probe that is placed over the lesion intraoperatively**.** All patients underwent a vitrectomy before radiotherapy and were treated with Bevacizumab before and after radiation. In a 4-minute exposure, the estimated dose at the lesion was 24 Gy**.** Results were excellent, with 76% of patients gaining at least 10 letters of visual acuity. Most of the responders required no more than one additional

**Integrins** as a mediator of adhesion both between cells and between cells and extracellular matrix, have a role in a variety of proliferative processes, including fibrosis. Integrin also seems to have a direct influence on proliferative kinase signalingare, a mediator of adhesion between cells and extracellular matrix. It is a transmembrane protein that binds to extracellular matrix proteins (fibronectin) allowing cell adhesion and cytoskeletal organization. The α5β1 is especially important in pathologic angiogenesis (not in normal vasculature) (Kim S et al., 2000). Many of the cellular effects of VEGF are duplicated downstream in the angiogenic cascade by the interaction between the transmembrane integrin α5β1receptor and its natural ligand, fibronectin. In addition to VEGF, the interaction of integrin α5β1 with fibronectin is critical to endothelial cell survival. Integrin α5β1 has been shown to be upregulated in all the cells associated with AMD pathogenesis, including endothelial cells, retinal pigment epithelium cells, macrophages, and fibroblasts. This implicates this molecule in multiple pathogenic processes involved in AMD, including neovascularization, vascular leakage, and inflammation (Klatt K et al., 2007). Two studies have demonstrated that once neovascular tissue begins to grow, the extracellular matrix needs to adhere to the neovascular endothelial cells for them to survive (Hynes RO, 2002; Hodivala-Dilke et al., 2003). Inhibiting the ligation of integrin α5β1 and fibronectin may disrupt the process of neovascularization, regardless of the upstream growth factor pathway. **Volociximab** (M200), a chimeric monoclonal antibody targeting integrin α5β1 to block its ligation of fibronectin, has robustly inhibited human umbilical vein endothelial cell tube formation in laboratory tests. It does so regardless of an initial growth factor stimulant. It has also inhibited neovascularization in primate choroid tissue and tumor angiogenesis in

**Vascular disrupting agents** also have potential for the inhibition of fibrosis as well as formation of new blood vessels. Unlike antiangiogenesis agents that block formation of new blood vessels, vascular-disrupting agents attack newly formed endothelium by disrupting connectivity between cells. This activity is expected to be complementary to anti-VEGF agents because it takes place at a later stage of neovascularization. It may also exert an important antifibrotic effect. A vascular-disrupting agent called **combretastatin** A4P (CA4P) has been evaluated in a Phase 1 study in humans with myopic macular degeneration, where it demonstrated relatively modest effects, but the characteristics of AMD may be more

Compared with age-matched controls, individuals with AMD demonstrate elevations in a variety of systemic biomarkers of inflammation, including activated monocytes and interleukin-6 (Vine AK et al., 2005 & Seddon et al., 2005). An increased risk of AMD in individuals with polymorphisms in their genes coding for the complement regulatory proteins is another signal that complement driven inflammation is perhaps an important mediator of this disease (Klein RJ et al., 2005). Although terminal elements of the complement pathway are

injection of Bevacizumab over the first year of follow-up.

rabbits (Ramakrishnan V et al., 2006; Bhaskar V et al., 2008).

suitable for its activity.

The potential for a single therapy to control the complex process of AMD seems to be relatively remote. It is not clear that a combination of different agents, working on the same pathway of extracellular VEGF inhibition, will reduce AMD progression, but there may be a strong potential for additive or synergistic effects from combining drugs with independent mechanisms. The current strategy of blocking VEGF in the extracellular space may be an inadequate approach for long-term control of AMD. Combining drugs that act at different points of the angiogenesis pathway has the potential to build on the benefits of extracellular VEGF inhibitors, but a more profound inhibition of the disease process may require activity in additional pathways of the disease.

#### **7. References**


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**10** 

*Hong Kong* 

**Nutritional Supplement Use and** 

Amy C. Y. Lo and Ian Y. Wong *Eye Institute, The University of Hong Kong* 

**Age-Related Macular Degeneration** 


Age-related macular degeneration (AMD) is a leading cause of irreversible visual impairment and blindness in the aging population 1. Yet, individuals with AMD have limited treatment options. Given the high prevalence and considerable public health

AMD is a multifactorial disease, with complex genetics and confounding environmental risk factors. The etiology of AMD still remains unknown, but oxidative stress to the retina and the retinal pigment epithelium (RPE) is one of the leading hypotheses in AMD

Oxidative stress and the cellular damages caused by reactive oxygen species (ROS) has been implicated in aging and age-related eye diseases 2. Most intracellular ROS are derived from the mitochondria in the electron transport chain. During fuel metabolism, oxygen consumption and ATP synthesis in the mitochondria, electrons are shuffled in sequential reduction and oxidation reactions in the electron transport chain. Yet, these reactions are not 100% efficient; electrons may "leak" out and result in the formation of ROS. ROS are highly reactive and unstable oxygen-containing atoms, ions, or molecules such as hydroxyl radical

"unpaired" electron in the outer shell, ROS are very unstable. In trying to achieve stability, ROS will then participate in further reduction and oxidation reactions, oxidizing target

Oxidative damages by ROS affect DNA and lipids inside the cell. Earlier senescence, which may be related to shortening of telomeric DNA, occurs after oxidative damage 3-6. Oxidative damage also results in point mutations and deletions in mitochondrial DNA 7. In fact, mitochondrial DNA is more susceptible to ROS-induced damage than nuclear DNA 8. As for lipids, ROS causes oxidation of lipid in a process called lipid peroxidation. The polyunsaturated fatty acids, a common and significant component of cell membrane, are particularly vulnerable to oxidation by ROS as a result of their many conjugated double bonds. Oxidation of polyunsaturated fatty acids results in the formation of reactive

molecules and resulting in generation of other free radicals by chain reaction.

aldehyde intermediates which are toxic to the cell 9.

burden, it is essential to understand the etiology and pathogenesis of AMD.

**1. Introduction** 

pathogenesis.

**2. Oxidative stress and AMD** 

(OH), superoxide anion (O2


## **Nutritional Supplement Use and Age-Related Macular Degeneration**

Amy C. Y. Lo and Ian Y. Wong *Eye Institute, The University of Hong Kong Hong Kong* 

#### **1. Introduction**

184 Age Related Macular Degeneration – The Recent Advances in Basic Research and Clinical Care

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degeneration. *Retina*, Vol. 26, No. 4, (April 2006), pp. 383–390, ISSN 0275-004X Spaide RF. (2009). Rationale for combination theray in age-related macular degeneration.

Spielberg L. & Leys A. (2010). Treatment of neovascular age-related macular degeneration

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Tatar O.; Shinoda K.; Kaiserling E.; Claes C. & Eckardt C. (2009). Implications of

Vine AK.; Stader J.; Branham K.; Musch DC. &, Swaroop A. (2005). Biomarkers of

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22, No. 1, (January 2003), pp. 1-29, ISSN 1350-9462

159-165, ISSN 1468-2079

2008), pp. 675-681, ISSN 0275-004X

bevacizumab combined with verteporfin photodynamic therapy for choroidal neovascularization in age-related macular degeneration. *Retina*, Vol. 28, No. 5, (may

treatment of choroidal neovascularization secondary to age- related macular

with a variable ranibizumab dosing regimen and one-time reduced-fluence photodynamic therapy: the TORPEDO trial at 2 years. *Graefes Arch Clin Exp* 

targeting VEGF effectively inhibits ocular neovascularization in a mouse model.

triamcinolone for therapy-resistant exudative age-related macular degeneration. *J Ocul Pharmacol Ther.* Vol. 26, No. 2, (April 2010), pp. 207-212, ISSN 1557-7732 Timke C.; Zieher H.; Roth A.; Hauser K. & Lipson KE. (2008). Combination of vascular

endothelial growth factor receptor/platelet derived growth factor receptor inhibition markedly improves radiation tumor therapy. *Clin Cancer Res*. Vol. 14,

bevacizumab on vascular endothelial growth factor and endostatin in human choroidal neovascularisation. *Br J Ophthalmol,* Vol. 93, No. 2, (February 2009), pp.

cardiovascular disease as risk factors for age-related macular degeneration. *Ophthalmology*, Vol. 112, No. 12, (December 2005), pp. 2076–2080, ISSN 0161-6420 Wan MJ.; Hooper PL. & Sheidow TG. (2010). Combination therapy in exudative age-related

macular degeneration: visual outcomes following combined treatment with photodynamic therapy and intravitreal bevacizumab. *Can J Ophthalmol*. Vol. 45, No.

endothelial growth factors and angiogenesis in eye disease. *Prog Retin Eye Res*. Vol.

Age-related macular degeneration (AMD) is a leading cause of irreversible visual impairment and blindness in the aging population 1. Yet, individuals with AMD have limited treatment options. Given the high prevalence and considerable public health burden, it is essential to understand the etiology and pathogenesis of AMD.

AMD is a multifactorial disease, with complex genetics and confounding environmental risk factors. The etiology of AMD still remains unknown, but oxidative stress to the retina and the retinal pigment epithelium (RPE) is one of the leading hypotheses in AMD pathogenesis.

#### **2. Oxidative stress and AMD**

Oxidative stress and the cellular damages caused by reactive oxygen species (ROS) has been implicated in aging and age-related eye diseases 2. Most intracellular ROS are derived from the mitochondria in the electron transport chain. During fuel metabolism, oxygen consumption and ATP synthesis in the mitochondria, electrons are shuffled in sequential reduction and oxidation reactions in the electron transport chain. Yet, these reactions are not 100% efficient; electrons may "leak" out and result in the formation of ROS. ROS are highly reactive and unstable oxygen-containing atoms, ions, or molecules such as hydroxyl radical (OH), superoxide anion (O2-) and hydrogen peroxide (H2O2). Due to the presence of the "unpaired" electron in the outer shell, ROS are very unstable. In trying to achieve stability, ROS will then participate in further reduction and oxidation reactions, oxidizing target molecules and resulting in generation of other free radicals by chain reaction.

Oxidative damages by ROS affect DNA and lipids inside the cell. Earlier senescence, which may be related to shortening of telomeric DNA, occurs after oxidative damage 3-6. Oxidative damage also results in point mutations and deletions in mitochondrial DNA 7. In fact, mitochondrial DNA is more susceptible to ROS-induced damage than nuclear DNA 8. As for lipids, ROS causes oxidation of lipid in a process called lipid peroxidation. The polyunsaturated fatty acids, a common and significant component of cell membrane, are particularly vulnerable to oxidation by ROS as a result of their many conjugated double bonds. Oxidation of polyunsaturated fatty acids results in the formation of reactive aldehyde intermediates which are toxic to the cell 9.

Nutritional Supplement Use and Age-Related Macular Degeneration 187

A summary of studies investigating the effect of nutritional supplements on the prevention

**investigated** 

beta-carotene; vitamin E

folic acid/B6/B12

alpha-carotene; betacarotene; beta cryptoxanthin; lutein + zeaxanthin; lycopene; vitamin E; zinc; fruit and vegetables; supplements

Eye Study (BMES) dietary fat, fish Australia (3654; ≥<sup>49</sup>

zinc

Dietary fat

**participants** 

vitamin E Australia (1,193; 55-

Finland (941; ≥65 years old)

80 years old)

Female health care professionals in USA

(5,442; ≥40 years )

(39,876; ≥45 years)

USA (1,709; 43-84 years old)

years old)

Health professionals in USA (104,208: 66,572 women, 37,636 men; ≥50 years old)

Health professionals in USA (72,489: 42,743 women, 29,746 men; ≥50 years old)

years old)

Dietary fat USA (11,448; 40-79

Female health professionals in USA

lutein 126 AMD patients 6-month

**(number; age) Follow-up** 

6-year prevalence

4-year incidence

Av 7.3 years

Av 10 years follow up

follow up

5-year incidence

8-10 year incidence

and progression of AMD is shown in Tables 1 and 2.

Alpha-tocopherol and beta-carotene study

Vitamin E, cataract, and age related maculopathy trial (VECAT)

Lutein Intervention Study Austria (LISA)

Nurses' Health Study (NHS) and Health Professional Followup Study (HPFS)

Nurses' Health Study (NHS) and Health Professional Followup Study (HPFS)

Third National Health and Nutrition Examination Study (NHANES III)

(ATBC)

Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS)

Christen 2010 75 Women's Health

et al 23 Beaver Dam study

Smith 2000 107 The Blue Mountains

*Randomized trials*

Teikari 1998 43

Taylor 2002 74

Christen 2009 132

Weigert 2011 61

*Population-based studies*

VandenLangenberg

Cho 2001 88

Cho 2001 108

Heuberger 2001 145

**Study Nutrients** 

Study (WHS) vitamin E

The retina is a structure that is particularly susceptible to oxidative damage by ROS. Firstly, the retina has the highest oxygen consumption in the body 10. In addition, constant exposure to incoming light in the retina can lead to photo-oxidation. The high oxygen consumption and high light exposure in the retina may in turn generate ROS. Moreover, the retina has a high lipid content, with abundant polyunsaturated fatty acids in the photoreceptor outer segments which are most prone to lipid peroxidation. In the neighborhood of photoreceptors are the RPE cells. Besides providing metabolic support to the photoreceptors, they also phagocytose the constantly shed parts of the photoreceptor outer segments. All these factors contribute to the susceptibility of the retina and RPE to oxidative stress.

With age, the susceptibility to oxidative damage in the retina increases. Aged rat retina showed decreased GSH-Px and catalase activities, which are related to increased lipid peroxidation with age 11. In particular, RPE cells accumulate lipofuscin granules during life. Lipofuscin granules are lysosomal residual bodies containing undigested end products from phagocytosis of photoreceptor outer segments 12. It was estimated that lipofuscin can occupy up to 19% of RPE cytoplasmic volume by the age of 80 when compared with only 1 % during the first decade of life 13. Lipofuscin has been shown to contain toxic substances, such as retinoids (products of the visual cycle) and oxidized proteins 14. Lipofuscin was also able to reduce RPE lysosomal and antioxidant activity 15. *In vitro* studies using porcine RPE cells showed that visible light irradiation can degrade RPE melanosomes, reduce melanin amount and increase ROS production, changes that also occur in human RPE melanosomes with aging 16.

#### **3. Nutritional supplements and AMD**

Oxidative stress has a recognized role in aging and AMD; treatments for AMD are therefore aimed at reducing oxidative stress-induced damage within the retina and RPE cells. This can be approached in two ways, either by decreasing the source of oxidative stress or by increasing the defense against oxidative stress. Among them, a tempting measure in lowering oxidative damage would be by dietary antioxidant supplementation. Data from observational studies have supported a link between nutritional factors with antioxidant properties and risks of AMD 17,18. Carotenoids, vitamin C and vitamin E with their antioxidant properties have been identified as having a potentially protective role. Other nutrients such as zinc and omega-3 fatty acids have been shown to be associated with reduced risk of AMD. Recently, B vitamins (folic acid, B6 and B12) have also been proposed to provide protection by a non-oxidative mechanism. Another nutritional supplement that has gained interest recently is the extracts from a group of fruit, berries.

Common nutrition supplements include:


The retina is a structure that is particularly susceptible to oxidative damage by ROS. Firstly, the retina has the highest oxygen consumption in the body 10. In addition, constant exposure to incoming light in the retina can lead to photo-oxidation. The high oxygen consumption and high light exposure in the retina may in turn generate ROS. Moreover, the retina has a high lipid content, with abundant polyunsaturated fatty acids in the photoreceptor outer segments which are most prone to lipid peroxidation. In the neighborhood of photoreceptors are the RPE cells. Besides providing metabolic support to the photoreceptors, they also phagocytose the constantly shed parts of the photoreceptor outer segments. All these factors contribute to

With age, the susceptibility to oxidative damage in the retina increases. Aged rat retina showed decreased GSH-Px and catalase activities, which are related to increased lipid peroxidation with age 11. In particular, RPE cells accumulate lipofuscin granules during life. Lipofuscin granules are lysosomal residual bodies containing undigested end products from phagocytosis of photoreceptor outer segments 12. It was estimated that lipofuscin can occupy up to 19% of RPE cytoplasmic volume by the age of 80 when compared with only 1 % during the first decade of life 13. Lipofuscin has been shown to contain toxic substances, such as retinoids (products of the visual cycle) and oxidized proteins 14. Lipofuscin was also able to reduce RPE lysosomal and antioxidant activity 15. *In vitro* studies using porcine RPE cells showed that visible light irradiation can degrade RPE melanosomes, reduce melanin amount and increase ROS production, changes that also occur in human RPE melanosomes

Oxidative stress has a recognized role in aging and AMD; treatments for AMD are therefore aimed at reducing oxidative stress-induced damage within the retina and RPE cells. This can be approached in two ways, either by decreasing the source of oxidative stress or by increasing the defense against oxidative stress. Among them, a tempting measure in lowering oxidative damage would be by dietary antioxidant supplementation. Data from observational studies have supported a link between nutritional factors with antioxidant properties and risks of AMD 17,18. Carotenoids, vitamin C and vitamin E with their antioxidant properties have been identified as having a potentially protective role. Other nutrients such as zinc and omega-3 fatty acids have been shown to be associated with reduced risk of AMD. Recently, B vitamins (folic acid, B6 and B12) have also been proposed to provide protection by a non-oxidative mechanism. Another nutritional supplement that

has gained interest recently is the extracts from a group of fruit, berries.

the susceptibility of the retina and RPE to oxidative stress.

**3. Nutritional supplements and AMD** 

Common nutrition supplements include: 1. AREDS and AREDS-type formulation

3. Vitamin C (L-ascorbic acid) 4. Vitamin E (α-tocopherol))

5. Zinc

7. B vitamins 8. Berry extracts

2. Carotenoids (β-carotene, lutein and zeaxanthin)

6. Omega-3 Long chain polyunsaturated fatty acids

with aging 16.

A summary of studies investigating the effect of nutritional supplements on the prevention and progression of AMD is shown in Tables 1 and 2.


Nutritional Supplement Use and Age-Related Macular Degeneration 189

**participants** 

USA (2,152; 45-84 years old)

USA (876; *CFH* and *LOC387715/ARMS2* genotype)

USA (681 twins; male only)

USA (4,519; 60-80 years old)

USA (4,003)

**(number; age)** 

old)

old)

lutein Austria (126 AMD

USA (151: 56 men, 95 women; 42-89 years

Austria (112: 48 men, 64 women; ≥50 years

USA (3640, 56% women; average 69

USA (90: 86 men, 4

women) 12 months

patients) 6 months

years old)

years old)

**Treatment duration** 

12-24 months

24 months

6 years

**(number; age) Follow-up** 

**investigated** 

omega-3 fatty acids;

omega-3 fatty acids;

total fat; saturated fat; oleate; linoleate; cholesterol; seafood

fish

fish

zinc

**Study Nutrients investigated participants** 

beta-carotene;vitamin C; vitamin E; copper;

/antioxidants/vitamins and minerals broad

zinc

lutein

spectrum supplementation formula

Table 2. Studies investigating nutritional supplements in the progression of AMD

Seddon 2001 109 Dietary fat, fish USA (349; 55-80

2008 115 AREDS omega-3 fatty acid; fish USA (2,132)

Table 1. Studies investigating nutritional supplements in the prevention of AMD

DHA; EPA; lutein/zeaxanthin; vitamin C; vitamin E;

DHA;

**Study Nutrients** 

Beaver Dam Study and Nutritional Factors in Eye Disease Study

Klein 2008 90 AREDS AREDS, zinc

US Twin Study of Age-Related Macular Degeneration

*Retrospective study*

Mares-Perlman

*Case controlled study*

Seddon 2006 111

SanGiovanni 2007

*Cross-sectional study*

AREDS 19

Richer 2004 55

SanGiovanni

Weigert 2011 61

<sup>113</sup> AREDS

Chiu 2009 146 AREDS

Newsome 1988 24 zinc

Stur 1996 87 Zinc

Veterans LAST study (Lutein Antioxidant Supplementation Trial)LAST

Lutein Intervention Study Austria (LISA)

1995


**participants** 

years old)

Health professionals in USA (118,428; ≥50 years old)

Australia (1,989; ≥49

Netherlands (4,170; ≥55 years old)

Australia (3654; ≥49

Women's Health Initiative (1,787; 50 to 79 years old), women only

France (832; ≥70 years old)

(113,058: 71,494 women and 41,564 men; ≥50 years old)

Australia (1,881; ≥49 years old; *CFH*  genotype)

Australia (2,454; ≥49

Australia (3654; ≥49

Netherlands (2,167; ≥55 years old; *CFH*

*LOC387715/ARMS2*

years old)

years old)

genotype)

and

years old)

**(number; age) Follow-up** 

5-year incidence

12-18 year incidence

Mean 8-year follow-up

5-year incidence

6 year prevalence

Up to 16 years in men, Up to 18 years in women

Mean 5.1 years and 10.5 years

Mean 8.6 years

10 year

10-year incidence

**investigated** 

alpha-carotene; betacarotene; beta cryptoxanthin; lutein + zeaxanthin; lycopene; vitamin A; vitamin C; zinc; supplements

alpha-carotene; betacarotene; beta cryptoxanthin; lutein + zeaxanthin; lycopene; vitamin A; vitamin C; vitamin E; fruits and vegetables; supplements

alpha-carotene; betacarotene; beta cryptoxanthin; lutein + zeaxanthin; lycopene; vitamin A; vitamin C; vitamin E; zinc

omega-3 fatty acid;

lutein + zeaxanthin; fruit and vegetable

total fish; white fish;

lutein + zeaxanthin

alpha-carotene; betacarotene; beta cryptoxanthin; lutein + zeaxanthin; lycopene; vitamins A; vitamin C ; vitamin E; iron; zinc

omega-3 fatty acid;

beta-carotene; lutein/zeaxanthin; DHA; EPA; zinc;

fish

fatty fish

Augood 2008 114 EUREYE DHA; EPA; oily fish Europe (4,753)

fish

**Study Nutrients** 

Flood 2002 44 The Blue Mountains

<sup>42</sup> Rotterdam Eye Study

Chua 2006 110 The Blue Mountains

Cho, 2004 52

van Leeuwen 2005

Moeller 2006 53

Delcourt 2007 112

Cho 2008 54

Eye Study (BMES)

Nurses' Health Study and men in the Health Professionals Follow-up Study

Eye Study (BMES)

Pathologies Oculaires

Nurses' health Study

Professionals Follow-

Eye Study (BMES)

Eye Study (BMES)

Eye Study (BMES) fish

Carotenoids in Age-related Eye Disease Study (CAREDS)

Liees a IAge (POLANUT)

and Health

up Study

Wang 2008 117 The Blue Mountains

Tan 2008 45 The Blue Mountains

Tan 2009 116 The Blue Mountains

Ho 2011 91 The Rotterdam Study


#### Table 1. Studies investigating nutritional supplements in the prevention of AMD


Table 2. Studies investigating nutritional supplements in the progression of AMD

Nutritional Supplement Use and Age-Related Macular Degeneration 191

beta-carotene) may be associated with an increased risk of lung cancer in smokers; vitamin C may cause renal stones; vitamin E may be associated with increased risk of hemorrhagic stroke; zinc can cause anemia, stomach upset, and may reduce serum high-density

Category 1 No AMD in both eyes <5 small drusen in one or

in one or both eyes

AMD in both eyes

Table 4. Categorization of AMD according to AREDS guidelines

formula to reducing the risk of development of advanced AMD.

Category 2 Mild to borderline AMD

Category 3 Absence of advanced

Category 4 Advanced AMD in one eye

outweigh the potential benefit in AMD protection.

hyperpigmentation or depigmentation 27

Brief description Clinical features Visual acuity

Multiple small or intermediate drusen in one or both eyes Pigment abnormalities in one or both eyes

Intermediate or large

Geographic atrophy Features not involving central macula

Advanced AMD or geographic atrophy in

No such features in better

20/32 or better in both

20/32 or better in both

20/32 or better in better

20/32 or better in better

eyes

eyes

eye

eye

both eyes

drusen

worse eye

eye

However, observations from the AREDS cohort failed to show any statistically significant serious side effects as mentioned above. Documented minor side effects included 1) increased genitourinary symptoms; 2) increased self-reported anemia; and 3) yellow discoloration of skin due to high level of vitamin A. Self-reported anemia was not correlated with any genuine reduction in blood hematocrit level. Smokers in the AREDS were discouraged from smoking, therefore whether the risk of lung cancer was increased was not being addressed. However, this has already been confirmed in two other trials 29,30. Hence, all smokers should be discouraged from smoking before the commencement of the AREDS formula. If he or she is not willing to quit smoking, the risk of having lung cancer may

In general, the AREDS formula was deemed safe and effective, in selected high-risk individuals 31. Inadequacy of the AREDS formula was that it did not include other potential ingredients such as lutein, zeaxanthin, and omega-3 fatty acid, which are also of particular interest due to their antioxidant abilities. In view of this, the National Eye Institute has launched the Age-Related Eye Disease Study 2 (AREDS2) in 2006, in hope to fill up the knowledge in this gap 32,33. In the AREDS2 formula, lutein, zeaxanthin, and omega-3 fatty acid have been added to the existing AREDS formula, and vitamin A was removed, mainly due to the potential risk associated with lung cancer. Results of the AREDS2 are expected to be available in 2012. Until then, the AREDS formula remains the only evidenced-based

Key: small drusen, <63 um in diameter (disc diameter around 1500 um); intermediate drusen, 63-124 um in diameter; large drusen, >125 um in diameter; pigment abnormalities refer to either

lipoprotein level.

#### **3.1 AREDS and AREDS-type formulation**

The Age-Related Eye Disease Study (AREDS) was a clinical trial sponsored by the National Eye Institute 19. This was to date the largest prospective randomized controlled trial to investigate the effect of an active supplement formula on the risk of development of AMD. The dosages of the supplements were at a high-than-normal level, because it was considered a form of active treatment, instead of a simple supplement pill. There were a total of 3,640 subjects, being monitored for an average of 6.3 years. Each subject was given either the AREDS formula, or placebo, to be taken on a twice-daily basis. Main components of the AREDS formula are vitamin A, vitamin C, vitamin E, and zinc. These were chosen because of their anti-oxidative abilities 20-25. When compared with the Dietary Reference Intake (DRI) issued by the Institute of Medicine, US National Academy, the dosage of ingredients in the AREDS formula was at a much higher level 26. For instance, the dosage of vitamin C in the AREDS formula was 500 mg/day, while that of the DRI was only 90mg per day. As far as vitamin C was concerned, one has to take at least 7 to 8 oranges per day, just to match up with what is provided by the AREDS pill 27. A comparison of the dosage in AREDS formulation with common fruits is given in Table 3.


@ Vitamin A as beta-carotene

\* Dietary Reference Intakes from the Institute of Medicine

\*\* 2.0-2.3 mg/day for men and 1.7-2.0 mg/day for women in United States

(Food and Nutrition Board, 2001)
