Nutrient contents of common fruits are expressed per 100 grams

Vitamin A@ International

@ Vitamin A as beta-carotene

category 3 or 4).

(Food and Nutrition Board, 2001)

comparison to common fruit items

microgram

Vitamin C milligram

Lutein + Zeaxanthin 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 lipoprotein level.


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 hyperpigmentation or depigmentation 27

Table 4. Categorization of AMD according to AREDS guidelines

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 outweigh the potential benefit in AMD protection.

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 formula to reducing the risk of development of advanced AMD.

Nutritional Supplement Use and Age-Related Macular Degeneration 193

population after 10-year of follow-up showed some interesting results. Instead of showing no effect of beta-carotene in AMD, Tan *et al* actually reported an increased risk of neovascular AMD with increasing beta-carotene intake 45. The authors found that increasing beta-carotene intake, either from diet alone or diet plus supplementation, was associated with higher risk neovascular AMD. This association also existed when the smoking status of

In fact, one has to bear in mind about the possible harmful effect of beta-carotene supplementation. Apart from the skin coloration, changes in scotopic b-wave during electroretinography and crystal formation have also been shown with long-term betacarotene use 46. More importantly, daily supplementation of beta-carotene in smokers was associated with a higher mortality rate due to ischemic heart disease and lung cancer 29,30. Since smoking also increases the risk of AMD, beta-carotene supplementation should be avoided in smokers. Currently, no biological explanation has been offered to clarify the

Lutein and zeaxanthin are the only two carotenoids that exist in the human retina 34,35. They are particularly dense in the macula in humans, where they are referred to as macular pigment 34. Macular pigment is thought to be protective against retinal damage. Three casecontrolled studies showed that there was an inverse association between the macular pigment density in the human retina and the risk of AMD 47-49. In an early study investigating the effects of high dietary carotenoid intake, lutein and zeaxanthin were found to be the specific carotenoids that are most strongly associated with reduced risk of AMD 20. This result was also supported by two other studies. The population-based Pathologies Oculaires Liees a l'Age (POLA) Study measured the plasma carotenoid levels by highperformance liquid chromatography (HPLC) in 899 subjects and correlated them with the risk of AMD 50. It was shown that high plasma levels of lutein and zeaxanthin were associated with a significant reduced risk of AMD. Similarly, a study in U.K. involving men and women aged 66 to 75 found that subjects with the lowest plasma level of zeaxanthin has a two-fold increased risk when compared with those with the highest plasma zeaxanthin,

Other studies also provide evidence in the association of lutein and zeaxanthin with AMD risk. In the Blue Mountains Eye Study, Flood *et al* reported a possible association between baseline intake of lutein and zeaxanthin and the 5-year incidence of early AMD 44. A longer, 10-year follow-up study reported that high dietary lutein and zeaxanthin intake (top tertile) was associated reduced risk of incident neovascular AMD 45. Participants with above

Conversely, several studies showed different results on the association of lutein and zeaxanthin. An early study in Beaver Dam (Beaver Dam Eye Study) reported no significant association between lutein and zeaxanthin and the risk of large drusen when 1,709 participants were followed up for 5 years 23. In a prospective follow-up study of women in the Nurses' Health Study and men in the Health Professionals Follow-up Study, Cho *et al* followed 77,562 women and 40,866 men ≥50 years old for up to 18 years for women and up to 12 years for men. It was reported that lutein and zeaxanthin were not strongly related to either early or neovascular AMD risk 52. The Carotenoids in Age-related Eye Disease Study (CAREDS), an ancillary study of the Women's Health Initiative, followed 1,787 female

the individuals was adjusted.

harmful effect of beta-carotene in human.

supporting the view that zeaxanthin may protect against AMD 51.

median intakes had a reduced risk of indistinct soft or reticular drusen.

#### **3.2 Carotenoids (**β**-carotene, lutein and zeaxanthin)**

Carotenoids are organic pigments naturally occurring in plants as well as in some algae, fungus and bacteria. Animals generally cannot synthesize carotenoids; they have to obtain carotenoids in their diet. There are two classes of carotenoids, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen) accounting for over 600 known carotenoids. A well known carotene is beta-carotene, the pigment that makes carrots orange. Interestingly, there are only two carotenoids that are present in the human retina 34,35, namely lutein [(3R,3'R,6'R)-beta,epsilon-Carotene-3,3'-diol] and its stereoisomer, zeaxanthin [(3R,3'R)-beta,beta-Carotene-3,3'-diol]. These carotenoids are enriched in the macula in high concentrations, thus giving the macula its yellowish color.

In human, four carotenoids including beta-carotene, alpha-carotene, gamma-carotene, and beta-cryptoxanthin can be converted into retinal, which is an important molecule in the photo-transduction pathway and therefore vision. Carotenoids can also absorb light and act as antioxidants by scavenging ROS such as . O2 and peroxyl radicals 36. In particular, two xanthophylls, lutein and zeaxanthin, have been shown to absorb the damaging blue light 36 as well as protect the retina 37 and retinal ganglion cells 38 from oxidative damage *in vitro*. In animal studies, lutein protected the inner retina against acute retinal ischemia/reperfusion injury due to its antioxidant properties 39.

Due to their antioxidant properties and blue light-filtering effects, the association of carotenoids with risk of AMD was explored. There have been conflicting results. Decreased risk of neovascular AMD has been found to be associated with higher levels of carotenoids in the serum samples 40. In monkeys, feeding a xanphophyll-free diet has been shown to promote drusen formation 41. In an early study based on National Health and Nutrition Examination Survey I data, an inverse association between the consumption of fruits and vegetables rich in pro-vitamin A carotenoids and the prevalence of AMD was demonstrated 22. In the Beaver Dam Eye Study, VandenLangenberg *et al* also found a significant but modest inverse association between intake of pro-vitamin A carotenoids and the incidence of large drusen 23. Later studies using the AREDS formulation suggested a beneficial effect of beta-carotene 19. The Rotterdam population-based study also reported a high dietary intake of beta-carotene together with vitamins C and E and zinc reduced the risk of AMD in elderly individuals 42. A 35% reduced risk of AMD was observed when an above-median intake of these 4 nutrients was given.

On the other hand, opposing results were obtained from other clinical trials and populationbased studies. The Alpha-Tocopherol and Beta-Carotene (ATBC) Study in Finland assessed the involvement of beta-carotene in occurrence of AMD among smoking males 43. Over 29,000 smoking males aged 50 to 69 years were given alpha-tocopherol (50 mg/day), betacarotene (20 mg/day), both of these, or placebo randomly. After 5 to 8 years of supplementation, Teikari *et al* found no beneficial effect of long-term beta-carotene supplementation on the incidence of AMD. The Blue Mountains Eye Study also reported no associations between beta-carotene intake and 5-year incidence of AMD 44. This is a population-based study including 1,989 individuals who finished a food frequency questionnaire. This questionnaire assessed the baseline intake of nutrients including alphacarotene, beta-carotene, beta-cryptoxanthin, lutein and zeaxanthin, lycopene, retinol, vitamin A, vitamin C, and zinc. For beta-carotene, Teikari *et al* suggested no evidence of protection by beta-carotene on the 5-year incidence of AMD. Further studies in the same

Carotenoids are organic pigments naturally occurring in plants as well as in some algae, fungus and bacteria. Animals generally cannot synthesize carotenoids; they have to obtain carotenoids in their diet. There are two classes of carotenoids, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen) accounting for over 600 known carotenoids. A well known carotene is beta-carotene, the pigment that makes carrots orange. Interestingly, there are only two carotenoids that are present in the human retina 34,35, namely lutein [(3R,3'R,6'R)-beta,epsilon-Carotene-3,3'-diol] and its stereoisomer, zeaxanthin [(3R,3'R)-beta,beta-Carotene-3,3'-diol]. These carotenoids are enriched in the macula in high concentrations, thus giving the macula its yellowish color. In human, four carotenoids including beta-carotene, alpha-carotene, gamma-carotene, and beta-cryptoxanthin can be converted into retinal, which is an important molecule in the photo-transduction pathway and therefore vision. Carotenoids can also absorb light and act

xanthophylls, lutein and zeaxanthin, have been shown to absorb the damaging blue light 36 as well as protect the retina 37 and retinal ganglion cells 38 from oxidative damage *in vitro*. In animal studies, lutein protected the inner retina against acute retinal ischemia/reperfusion

Due to their antioxidant properties and blue light-filtering effects, the association of carotenoids with risk of AMD was explored. There have been conflicting results. Decreased risk of neovascular AMD has been found to be associated with higher levels of carotenoids in the serum samples 40. In monkeys, feeding a xanphophyll-free diet has been shown to promote drusen formation 41. In an early study based on National Health and Nutrition Examination Survey I data, an inverse association between the consumption of fruits and vegetables rich in pro-vitamin A carotenoids and the prevalence of AMD was demonstrated 22. In the Beaver Dam Eye Study, VandenLangenberg *et al* also found a significant but modest inverse association between intake of pro-vitamin A carotenoids and the incidence of large drusen 23. Later studies using the AREDS formulation suggested a beneficial effect of beta-carotene 19. The Rotterdam population-based study also reported a high dietary intake of beta-carotene together with vitamins C and E and zinc reduced the risk of AMD in elderly individuals 42. A 35% reduced risk of AMD was observed when an above-median

On the other hand, opposing results were obtained from other clinical trials and populationbased studies. The Alpha-Tocopherol and Beta-Carotene (ATBC) Study in Finland assessed the involvement of beta-carotene in occurrence of AMD among smoking males 43. Over 29,000 smoking males aged 50 to 69 years were given alpha-tocopherol (50 mg/day), betacarotene (20 mg/day), both of these, or placebo randomly. After 5 to 8 years of supplementation, Teikari *et al* found no beneficial effect of long-term beta-carotene supplementation on the incidence of AMD. The Blue Mountains Eye Study also reported no associations between beta-carotene intake and 5-year incidence of AMD 44. This is a population-based study including 1,989 individuals who finished a food frequency questionnaire. This questionnaire assessed the baseline intake of nutrients including alphacarotene, beta-carotene, beta-cryptoxanthin, lutein and zeaxanthin, lycopene, retinol, vitamin A, vitamin C, and zinc. For beta-carotene, Teikari *et al* suggested no evidence of protection by beta-carotene on the 5-year incidence of AMD. Further studies in the same

O2 and peroxyl radicals 36. In particular, two

**3.2 Carotenoids (**β**-carotene, lutein and zeaxanthin)** 

as antioxidants by scavenging ROS such as .

injury due to its antioxidant properties 39.

intake of these 4 nutrients was given.

population after 10-year of follow-up showed some interesting results. Instead of showing no effect of beta-carotene in AMD, Tan *et al* actually reported an increased risk of neovascular AMD with increasing beta-carotene intake 45. The authors found that increasing beta-carotene intake, either from diet alone or diet plus supplementation, was associated with higher risk neovascular AMD. This association also existed when the smoking status of the individuals was adjusted.

In fact, one has to bear in mind about the possible harmful effect of beta-carotene supplementation. Apart from the skin coloration, changes in scotopic b-wave during electroretinography and crystal formation have also been shown with long-term betacarotene use 46. More importantly, daily supplementation of beta-carotene in smokers was associated with a higher mortality rate due to ischemic heart disease and lung cancer 29,30. Since smoking also increases the risk of AMD, beta-carotene supplementation should be avoided in smokers. Currently, no biological explanation has been offered to clarify the harmful effect of beta-carotene in human.

Lutein and zeaxanthin are the only two carotenoids that exist in the human retina 34,35. They are particularly dense in the macula in humans, where they are referred to as macular pigment 34. Macular pigment is thought to be protective against retinal damage. Three casecontrolled studies showed that there was an inverse association between the macular pigment density in the human retina and the risk of AMD 47-49. In an early study investigating the effects of high dietary carotenoid intake, lutein and zeaxanthin were found to be the specific carotenoids that are most strongly associated with reduced risk of AMD 20. This result was also supported by two other studies. The population-based Pathologies Oculaires Liees a l'Age (POLA) Study measured the plasma carotenoid levels by highperformance liquid chromatography (HPLC) in 899 subjects and correlated them with the risk of AMD 50. It was shown that high plasma levels of lutein and zeaxanthin were associated with a significant reduced risk of AMD. Similarly, a study in U.K. involving men and women aged 66 to 75 found that subjects with the lowest plasma level of zeaxanthin has a two-fold increased risk when compared with those with the highest plasma zeaxanthin, supporting the view that zeaxanthin may protect against AMD 51.

Other studies also provide evidence in the association of lutein and zeaxanthin with AMD risk. In the Blue Mountains Eye Study, Flood *et al* reported a possible association between baseline intake of lutein and zeaxanthin and the 5-year incidence of early AMD 44. A longer, 10-year follow-up study reported that high dietary lutein and zeaxanthin intake (top tertile) was associated reduced risk of incident neovascular AMD 45. Participants with above median intakes had a reduced risk of indistinct soft or reticular drusen.

Conversely, several studies showed different results on the association of lutein and zeaxanthin. An early study in Beaver Dam (Beaver Dam Eye Study) reported no significant association between lutein and zeaxanthin and the risk of large drusen when 1,709 participants were followed up for 5 years 23. In a prospective follow-up study of women in the Nurses' Health Study and men in the Health Professionals Follow-up Study, Cho *et al* followed 77,562 women and 40,866 men ≥50 years old for up to 18 years for women and up to 12 years for men. It was reported that lutein and zeaxanthin were not strongly related to either early or neovascular AMD risk 52. The Carotenoids in Age-related Eye Disease Study (CAREDS), an ancillary study of the Women's Health Initiative, followed 1,787 female

Nutritional Supplement Use and Age-Related Macular Degeneration 195

supplementation with macular pigment optical density and visual acuity in 126 AMD patients randomly assigned to lutein supplementation or placebo 61. Weigert *et al* observed that lutein could significantly increase macular pigment optical density despite having no effect on mean differential light threshold or visual acuity. Interestingly, a significant correlation was found between the lutein-induced increase in macular pigment optical density and the change in mean differential light threshold and visual acuity. This finding suggests that patients who experience a pronounced increase in macular pigment optical

As lutein and zeaxanthin were not ready for manufacturing as a research formula, neither of them was included in the AREDS formula 28. The US Food and Drug Administration (FDA) has conducted an evidence-based review to evaluate the role of lutein and zeaxanthin in reducing the risk of AMD 62. After reviewing a number of intervention and observational studies, the FDA denied a health claim about the intake of lutein or zeaxanthin (or both) and the risk of AMD in 2006. However, in view of the conflicting findings, the National Eye Institute (Bethesda, Maryland, USA) launched the Age-Related Eye Disease Study 2 (AREDS2) in 2006, hoping to resolve the link between carotenoids (lutein and zeaxanthin) intake and AMD protection 32,33. The AREDS2, a large, multi-centered, randomized trial, is currently underway to address the effects of high dose lutein and zeaxanthin supplementation and/or omega-3 fatty acids on the progression of AMD. Beta-carotene, which increases the risk of lung cancer in smokers 29,30, is removed from the AREDS2 formula. Another on-going, similar randomized controlled trial is the Carotenoids in Age-Related Maculopathy (CARMA) Study 63. In this study, 433 participants with either early AMD features or any level of AMD in one eye and advanced AMD in the fellow eye were recruited. Either lutein and zeaxanthin, in combination with antioxidants (including vitamin C, vitamin E, zinc, and copper) or placebo was given. Again, beta-carotene was excluded in

Although the beneficial effects have not been proven, lutein and zeaxanthin are included in daily supplements and food additives and can be obtained over the counter. Moreover, the addition of crystalline lutein into food and beverage products is considered GRAS (generally recognized as safe) and is approved by the FDA 64. Lutein toxicity studies in animals using high doses of purified crystalline lutein revealed no unfavorable events 64 and no adverse events are reported for lutein and zeaxanthin at doses up to 40 mg/day in human for 2 months 65. The risk profile of lutein was also recently reviewed in 2006 by the Council for Responsible Nutrition (CRN) in Washington, D.C. It was concluded that apart from the reversible skin discoloration, no other adverse effects were observed 66. The CRN suggested an upper level of intake for lutein up to 20 mg/day. Currently, the average daily intake for lutein and zeaxanthin is 2.0-2.3 mg/day for men and 1.7-2.0 mg/day for women

In view of their potential benefits as well as minimal side effects, lutein and zeaxanthin may

Vitamin C is a water-soluble nutrient that is synthesized in almost all animals and plants. It is well known for its potent antioxidant activities 67,68. It also acts as an important co-factor

density after lutein supplementation may benefit in terms of visual function.

the preparation due to the increased risk of lung cancer in smokers 29,30.

in United States (Food and Nutrition Board, 2001).

**3.3 Vitamin C (L-ascorbic acid)** 

be recommended for those who are keen and at risk of AMD 27.

participants aged 50 to 79 for 4 to 7 years 53 and assessed their diet by a food frequency questionnaire. Subjects were divided according to their lutein and zeaxanthin intake, but there was no statistical difference between the amount of lutein and zeaxanthin intake and the prevalence of intermediate AMD. A later large prospective follow-up study also reported similar results 54. Two cohorts, the Nurses' Health Study and the Health Professionals Follow-up Study which included 51,564 men and 71,494 women aged ≥50 years were followed up for up to 18 years. Cho *et al* reported that there was no association between lutein/zeaxanthin intake and the risk of self-reported early AMD. Yet, a nonsignificant and nonlinear inverse association between lutein/zeaxanthin intake and neovascular AMD risk was observed.

More recently, lutein itself has gained special interests. Two prospective randomized controlled trials have investigated the association of lutein supplementation and the incidence of AMD. The larger Veterans LAST study (Lutein Antioxidant Supplementation Trial) involved 90 subjects with atrophic AMD who were randomly divided into three groups: lutein (10mg) group, lutein (10mg) plus additional antioxidants and nutrients group, and maltodextrin placebo group 55. Subjects were followed for 12 months and those who received lutein alone or lutein plus antioxidants and nutrients had improved visual acuity. Richer *et al* concluded that lutein alone or in combination with other nutritional supplements (including zinc, beta-carotene and vitamins C and E) is protective and slow down the progression of AMD. On the other hand, a smaller prospective trial measured the contrast sensitivity in 25 subjects after lutein supplementation (6mg) with vitamins and minerals or placebo over a 6-month period 56. No statistical difference was observed between the lutein and placebo group, suggesting no significant association between lutein supplementation and AMD. However, one has to be careful about these findings. The sample sizes in both studies were fairly small and the follow-up periods were limited to 12 months or less.

More supportive evidence came from a recent study in which participants in AREDS were genotyped for the hepatic lipase (*LIPC*) gene 57. Hepatic lipase is a protein in the highdensity lipoprotein cholesterol pathway and has been shown in a large genome-wide association study to be a novel locus for advanced AMD risk 58. It was observed in the AREDS participants that lower dietary lutein intake was significantly associated with increased risk of advance AMD, after controlling for the *LIPC* genotype. This suggests that high dietary lutein intake may reduce the risk of advanced AMD, after adjusting for genetic variants.

Lutein is also a macular pigment. Due to lutein's antioxidant properties and blue-light filtering capacity 36, it was hypothesized that macular pigment may provide protection against the development of AMD 59. The first prospective follow-up study, Muenster Aging and Retina Study (MARS), recently investigated the determinants of macular pigment optical density and its relation to AMD 60. Foveal macular pigment optical density was accessed in 369 participants including patients with different stages of AMD and healthy controls. In the 2.6-year follow-up study, it was observed that serum level of lutein, lutein supplementation in particular, was the strongest determinants of macular pigment optical density. However, the hypothetical protective effect of macular pigment in AMD could not be confirmed. On the other hand, a recent double-masked controlled study, Lutein Intervention Study Austria (LISA), investigated the association of 6-month lutein

participants aged 50 to 79 for 4 to 7 years 53 and assessed their diet by a food frequency questionnaire. Subjects were divided according to their lutein and zeaxanthin intake, but there was no statistical difference between the amount of lutein and zeaxanthin intake and the prevalence of intermediate AMD. A later large prospective follow-up study also reported similar results 54. Two cohorts, the Nurses' Health Study and the Health Professionals Follow-up Study which included 51,564 men and 71,494 women aged ≥50 years were followed up for up to 18 years. Cho *et al* reported that there was no association between lutein/zeaxanthin intake and the risk of self-reported early AMD. Yet, a nonsignificant and nonlinear inverse association between lutein/zeaxanthin intake and

More recently, lutein itself has gained special interests. Two prospective randomized controlled trials have investigated the association of lutein supplementation and the incidence of AMD. The larger Veterans LAST study (Lutein Antioxidant Supplementation Trial) involved 90 subjects with atrophic AMD who were randomly divided into three groups: lutein (10mg) group, lutein (10mg) plus additional antioxidants and nutrients group, and maltodextrin placebo group 55. Subjects were followed for 12 months and those who received lutein alone or lutein plus antioxidants and nutrients had improved visual acuity. Richer *et al* concluded that lutein alone or in combination with other nutritional supplements (including zinc, beta-carotene and vitamins C and E) is protective and slow down the progression of AMD. On the other hand, a smaller prospective trial measured the contrast sensitivity in 25 subjects after lutein supplementation (6mg) with vitamins and minerals or placebo over a 6-month period 56. No statistical difference was observed between the lutein and placebo group, suggesting no significant association between lutein supplementation and AMD. However, one has to be careful about these findings. The sample sizes in both studies were fairly small and the follow-up periods were limited to 12

More supportive evidence came from a recent study in which participants in AREDS were genotyped for the hepatic lipase (*LIPC*) gene 57. Hepatic lipase is a protein in the highdensity lipoprotein cholesterol pathway and has been shown in a large genome-wide association study to be a novel locus for advanced AMD risk 58. It was observed in the AREDS participants that lower dietary lutein intake was significantly associated with increased risk of advance AMD, after controlling for the *LIPC* genotype. This suggests that high dietary lutein intake may reduce the risk of advanced AMD, after adjusting for genetic

Lutein is also a macular pigment. Due to lutein's antioxidant properties and blue-light filtering capacity 36, it was hypothesized that macular pigment may provide protection against the development of AMD 59. The first prospective follow-up study, Muenster Aging and Retina Study (MARS), recently investigated the determinants of macular pigment optical density and its relation to AMD 60. Foveal macular pigment optical density was accessed in 369 participants including patients with different stages of AMD and healthy controls. In the 2.6-year follow-up study, it was observed that serum level of lutein, lutein supplementation in particular, was the strongest determinants of macular pigment optical density. However, the hypothetical protective effect of macular pigment in AMD could not be confirmed. On the other hand, a recent double-masked controlled study, Lutein Intervention Study Austria (LISA), investigated the association of 6-month lutein

neovascular AMD risk was observed.

months or less.

variants.

supplementation with macular pigment optical density and visual acuity in 126 AMD patients randomly assigned to lutein supplementation or placebo 61. Weigert *et al* observed that lutein could significantly increase macular pigment optical density despite having no effect on mean differential light threshold or visual acuity. Interestingly, a significant correlation was found between the lutein-induced increase in macular pigment optical density and the change in mean differential light threshold and visual acuity. This finding suggests that patients who experience a pronounced increase in macular pigment optical density after lutein supplementation may benefit in terms of visual function.

As lutein and zeaxanthin were not ready for manufacturing as a research formula, neither of them was included in the AREDS formula 28. The US Food and Drug Administration (FDA) has conducted an evidence-based review to evaluate the role of lutein and zeaxanthin in reducing the risk of AMD 62. After reviewing a number of intervention and observational studies, the FDA denied a health claim about the intake of lutein or zeaxanthin (or both) and the risk of AMD in 2006. However, in view of the conflicting findings, the National Eye Institute (Bethesda, Maryland, USA) launched the Age-Related Eye Disease Study 2 (AREDS2) in 2006, hoping to resolve the link between carotenoids (lutein and zeaxanthin) intake and AMD protection 32,33. The AREDS2, a large, multi-centered, randomized trial, is currently underway to address the effects of high dose lutein and zeaxanthin supplementation and/or omega-3 fatty acids on the progression of AMD. Beta-carotene, which increases the risk of lung cancer in smokers 29,30, is removed from the AREDS2 formula. Another on-going, similar randomized controlled trial is the Carotenoids in Age-Related Maculopathy (CARMA) Study 63. In this study, 433 participants with either early AMD features or any level of AMD in one eye and advanced AMD in the fellow eye were recruited. Either lutein and zeaxanthin, in combination with antioxidants (including vitamin C, vitamin E, zinc, and copper) or placebo was given. Again, beta-carotene was excluded in the preparation due to the increased risk of lung cancer in smokers 29,30.

Although the beneficial effects have not been proven, lutein and zeaxanthin are included in daily supplements and food additives and can be obtained over the counter. Moreover, the addition of crystalline lutein into food and beverage products is considered GRAS (generally recognized as safe) and is approved by the FDA 64. Lutein toxicity studies in animals using high doses of purified crystalline lutein revealed no unfavorable events 64 and no adverse events are reported for lutein and zeaxanthin at doses up to 40 mg/day in human for 2 months 65. The risk profile of lutein was also recently reviewed in 2006 by the Council for Responsible Nutrition (CRN) in Washington, D.C. It was concluded that apart from the reversible skin discoloration, no other adverse effects were observed 66. The CRN suggested an upper level of intake for lutein up to 20 mg/day. Currently, the average daily intake for lutein and zeaxanthin is 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).

In view of their potential benefits as well as minimal side effects, lutein and zeaxanthin may be recommended for those who are keen and at risk of AMD 27.

#### **3.3 Vitamin C (L-ascorbic acid)**

Vitamin C is a water-soluble nutrient that is synthesized in almost all animals and plants. It is well known for its potent antioxidant activities 67,68. It also acts as an important co-factor

Nutritional Supplement Use and Age-Related Macular Degeneration 197

More importantly, a negative association between vitamin E and AMD was recently reported. In the Blue Mountains Eye Study involving an Australian population–based cohort, Tan *et al* reported that high vitamin E intake was associated with increased risk of late AMD, suggesting a harmful effect of dietary vitamin E on risk of AMD 45. However, one has to be cautious about these results. There was a moderate loss of participants in this particular study, while the levels of vitamin E intake between participants followed up and not followed up were significantly different. The authors mentioned that this might affect

Zinc is an essential trace element for almost all organisms including plants, animals and microorganisms. It has a multitude of biological roles, playing a fundamental role in cellular metabolism. For example, it plays a structural role in a large number of transcription factors containing zinc fingers and similar structural motifs. Most importantly, it was first shown to be required for the catalytic activity of carbonic anhydrase 76. Later studies showed that zinc has a catalytic or structural role in at least 300 zinc metalloenzymes 77-79, influencing many metabolic reactions. In fact, approximately 10% of the human genome encodes for proteins

In the human body, there are about 2-3 g of zinc, making it the second most abundant trace element 79,81. In ocular tissues, the concentration of zinc is unusually high when compared with other tissues 82. In the eye zinc is most abundant in the retina and choroid, followed by ciliary body, iris, optic nerve, sclera, cornea, and lens 83. A number of functions of zinc in the retina have been suggested, including modulation of retinal synaptic transmission, modification of photoreceptor plasma membrane, involvement in retinal vitamin A metabolism, regulation of light-rhodopsin reaction within the photoreceptor, and

There are subtle ocular manifestations associated with zinc deficiency. In a prospective, randomized, double-masked, placebo-controlled investigation of the effects of oral zinc administration on the visual acuity outcome in 151 subjects with drusen or macular degeneration, the treatment group had significantly less visual loss than the placebo group 24. As elderly patients are found to be at higher risk of zinc deficiency 86, this may suggest an

For the past three decades, there have been considerable interest and controversy related to zinc supplementation in AMD patients. To date, results on zinc supplementation and AMD have been mixed. As described above, Newsome *et al* reported significant reduction in visual loss in AMD patients when supplemented with oral zinc 24. Moreover, Mares-Perlman *et al* reported a weak protective effect of dietary zinc on the development of some forms of early AMD 71. In the large double-masked clinical trial, The Age-Related Eye Disease Study (AREDS), involving 11 centers, participants taking zinc alone demonstrated an odds reduction of 0.75 for the development of advanced AMD. Zinc significantly reduced the odds of developing advanced AMD in the higher-risk group. A population-based cohort study reported that high dietary zinc intake was associated with a lower risk of incident AMD 42. In the Beaver Dam Eye Study, it was observed that there is a significant inverse association between zinc and the incidence of pigmentary abnormalities, but there was no

increased risk of vision loss from AMD in elderly patients.

the interpretation of the observed results.

**3.5 Zinc** 

that can bind zinc 80.

antioxidant activity 84,85.

in mammals as in the synthesis of collagen; therefore vitamin C is used in the treatment and prevention of scurvy. In ophthalmology, there has not been any randomized controlled trial in assessing the efficacy of vitamin C as a single supplement in AMD. Yet, in other studies combining vitamin C with other supplements, data on the protective effects of vitamin C has been mixed. Vitamin C is shown to be beneficial in the AREDS study 19. In two large prospective studies of 135 men and 329 women with up to 18 years of follow-up 52, it was found that higher fruit intake was related to a reduced risk of neovascular age-related maculopathy but none of the vitamins (including vitamin C) or carotenoids examined was clearly related to the disease. In a population-based cohort study involving 1,586 middleaged and older adults, the researchers found no significant associations between the risk of large drusen and intake of vitamin C 23. Another population-based cohort study even suggested that an increasing baseline vitamin C intake from diet and supplements was associated with an increased risk of incident early age-related maculopathy when compared with the lowest quintile 44

#### **3.4 Vitamin E (**α**-tocopherol)**

Vitamin E is a collective term for a group of natural lipid-soluble compounds containing the tocopherols (α-, β-, γ- and δ-) and tocotrienols (α-, β-, γ- and δ-) with antioxidant properties. Among them, α-tocopherol is the only form to meet human requirements. In the eye, αtocopherol can be found in the retina, RPE and choroid 69. Its concentration in the retina increases after oral supplements 70.

As an antioxidant and a nutritional factor, vitamin E has been explored in its association with prevention of AMD. Again, data for vitamin E have been mixed. Some studies reported that higher intake are associated with lower risks of AMD or signs 23,42,71 whereas some concluded no associations 45,52,72,73.

In particular, three large randomized controlled trials have assessed vitamin E in the incidence of AMD. The Alpha-Tocopherol and Beta-Carotene (ATBC) Study involved over 29,000 smoking males aged 50 to 69 years who were randomly assigned to alphatocopherol (50 mg/day), beta-carotene (20 mg/day), both of these, or placebo 43. Of these, an end-of-trial ophthalmological examination was performed in a random sample of 941 participants aged 65 years or more. No beneficial effect of long-term supplementation with alpha-tocopherol on the occurrence of AMD was detected among smoking males. In the Vitamin E Cataract and Age-related Maculopathy Trial (VECAT), 1,193 healthy volunteers aged between 55 and 80 years were randomly given either vitamin E (500IU = 335 mg) or placebo daily for 4 years 74. In the study, the incidence of early AMD in those receiving vitamin E (8.6%) was similar to those on placebo (8.1%) whereas for late disease the incidence was 0.8% versus 0.6%. Again, daily vitamin E supplement does not prevent the development or progression of early or later stages of AMD. In the Women's Health Study (WHS) 75, a large scale randomized trial of women, 39,876 healthy female health professionals were randomly assigned to receive with natural source vitamin E (600IU) or placebo on alternate days. There were 117 AMD cases in the vitamin E group versus 128 cases in the placebo group after 10 years of treatment and follow-up. Similar to other studies, no large beneficial or harmful effect on risk of AMD was observed in long term vitamin E supplementation.

More importantly, a negative association between vitamin E and AMD was recently reported. In the Blue Mountains Eye Study involving an Australian population–based cohort, Tan *et al* reported that high vitamin E intake was associated with increased risk of late AMD, suggesting a harmful effect of dietary vitamin E on risk of AMD 45. However, one has to be cautious about these results. There was a moderate loss of participants in this particular study, while the levels of vitamin E intake between participants followed up and not followed up were significantly different. The authors mentioned that this might affect the interpretation of the observed results.

#### **3.5 Zinc**

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

in mammals as in the synthesis of collagen; therefore vitamin C is used in the treatment and prevention of scurvy. In ophthalmology, there has not been any randomized controlled trial in assessing the efficacy of vitamin C as a single supplement in AMD. Yet, in other studies combining vitamin C with other supplements, data on the protective effects of vitamin C has been mixed. Vitamin C is shown to be beneficial in the AREDS study 19. In two large prospective studies of 135 men and 329 women with up to 18 years of follow-up 52, it was found that higher fruit intake was related to a reduced risk of neovascular age-related maculopathy but none of the vitamins (including vitamin C) or carotenoids examined was clearly related to the disease. In a population-based cohort study involving 1,586 middleaged and older adults, the researchers found no significant associations between the risk of large drusen and intake of vitamin C 23. Another population-based cohort study even suggested that an increasing baseline vitamin C intake from diet and supplements was associated with an increased risk of incident early age-related maculopathy when compared

Vitamin E is a collective term for a group of natural lipid-soluble compounds containing the tocopherols (α-, β-, γ- and δ-) and tocotrienols (α-, β-, γ- and δ-) with antioxidant properties. Among them, α-tocopherol is the only form to meet human requirements. In the eye, αtocopherol can be found in the retina, RPE and choroid 69. Its concentration in the retina

As an antioxidant and a nutritional factor, vitamin E has been explored in its association with prevention of AMD. Again, data for vitamin E have been mixed. Some studies reported that higher intake are associated with lower risks of AMD or signs 23,42,71 whereas some

In particular, three large randomized controlled trials have assessed vitamin E in the incidence of AMD. The Alpha-Tocopherol and Beta-Carotene (ATBC) Study involved over 29,000 smoking males aged 50 to 69 years who were randomly assigned to alphatocopherol (50 mg/day), beta-carotene (20 mg/day), both of these, or placebo 43. Of these, an end-of-trial ophthalmological examination was performed in a random sample of 941 participants aged 65 years or more. No beneficial effect of long-term supplementation with alpha-tocopherol on the occurrence of AMD was detected among smoking males. In the Vitamin E Cataract and Age-related Maculopathy Trial (VECAT), 1,193 healthy volunteers aged between 55 and 80 years were randomly given either vitamin E (500IU = 335 mg) or placebo daily for 4 years 74. In the study, the incidence of early AMD in those receiving vitamin E (8.6%) was similar to those on placebo (8.1%) whereas for late disease the incidence was 0.8% versus 0.6%. Again, daily vitamin E supplement does not prevent the development or progression of early or later stages of AMD. In the Women's Health Study (WHS) 75, a large scale randomized trial of women, 39,876 healthy female health professionals were randomly assigned to receive with natural source vitamin E (600IU) or placebo on alternate days. There were 117 AMD cases in the vitamin E group versus 128 cases in the placebo group after 10 years of treatment and follow-up. Similar to other studies, no large beneficial or harmful effect on risk of AMD was observed in long term

with the lowest quintile 44

**3.4 Vitamin E (**α**-tocopherol)** 

increases after oral supplements 70.

concluded no associations 45,52,72,73.

vitamin E supplementation.

Zinc is an essential trace element for almost all organisms including plants, animals and microorganisms. It has a multitude of biological roles, playing a fundamental role in cellular metabolism. For example, it plays a structural role in a large number of transcription factors containing zinc fingers and similar structural motifs. Most importantly, it was first shown to be required for the catalytic activity of carbonic anhydrase 76. Later studies showed that zinc has a catalytic or structural role in at least 300 zinc metalloenzymes 77-79, influencing many metabolic reactions. In fact, approximately 10% of the human genome encodes for proteins that can bind zinc 80.

In the human body, there are about 2-3 g of zinc, making it the second most abundant trace element 79,81. In ocular tissues, the concentration of zinc is unusually high when compared with other tissues 82. In the eye zinc is most abundant in the retina and choroid, followed by ciliary body, iris, optic nerve, sclera, cornea, and lens 83. A number of functions of zinc in the retina have been suggested, including modulation of retinal synaptic transmission, modification of photoreceptor plasma membrane, involvement in retinal vitamin A metabolism, regulation of light-rhodopsin reaction within the photoreceptor, and antioxidant activity 84,85.

There are subtle ocular manifestations associated with zinc deficiency. In a prospective, randomized, double-masked, placebo-controlled investigation of the effects of oral zinc administration on the visual acuity outcome in 151 subjects with drusen or macular degeneration, the treatment group had significantly less visual loss than the placebo group 24. As elderly patients are found to be at higher risk of zinc deficiency 86, this may suggest an increased risk of vision loss from AMD in elderly patients.

For the past three decades, there have been considerable interest and controversy related to zinc supplementation in AMD patients. To date, results on zinc supplementation and AMD have been mixed. As described above, Newsome *et al* reported significant reduction in visual loss in AMD patients when supplemented with oral zinc 24. Moreover, Mares-Perlman *et al* reported a weak protective effect of dietary zinc on the development of some forms of early AMD 71. In the large double-masked clinical trial, The Age-Related Eye Disease Study (AREDS), involving 11 centers, participants taking zinc alone demonstrated an odds reduction of 0.75 for the development of advanced AMD. Zinc significantly reduced the odds of developing advanced AMD in the higher-risk group. A population-based cohort study reported that high dietary zinc intake was associated with a lower risk of incident AMD 42. In the Beaver Dam Eye Study, it was observed that there is a significant inverse association between zinc and the incidence of pigmentary abnormalities, but there was no

Nutritional Supplement Use and Age-Related Macular Degeneration 199

treated participants withdrew from the studies due to gastrointestinal symptoms when

The retina contains abundant fatty acids, about 30% of which are polyunsaturated fatty acids 93. Polyunsaturated fatty acids are classified into 2 groups: ω-3 and ω-6 depending on the position of the first double bond from the methyl end of the molecule. Docosahexaenoic acid (DHA), an omega-3 fatty acid, is highly enriched in the retina, particularly in the disc membrane of photoreceptor outer segments 94. DHA is the major polyunsaturated fatty acid in cerebral gray matter as well. Yet, the specific role of DHA in the eye is not clear. DHA has been shown to be important for photoreceptor survival 95-98. DHA may have a role in modulating G protein-coupled signaling pathways that are involved in visual transduction 99. DHA may also affect rhodopsin function during photoreception by influencing the membrane's biophysical properties 100,101. In rhesus monkeys, dietary depletion of alphalinolenic acid, a dietary precursor of DHA, resulted in undetectable plasma DHA level and more importantly, abnormal retinogram and visual impairment 102,103. Nonetheless, DHA supplementation is effective in improving retinal function in a patient with autosomal dominant Stargardt-like retinal dystrophy 104. The importance of DHA in retinal function

may suggest a possible beneficial role of DHA in retinal disease such as AMD.

become attractive alternatives in lowering the risk of AMD.

may have decreased the observed AMD prevalence.

Another omega-3 fatty acid, eicosapentaenoic acid (EPA), is the precursor of eicosanoids in the body. It can act as a competitive inhibitor of arachidonic acid conversion to proinflammatory eicosanoids prostaglandin E(2) and leukotriene B(4) 105. As inflammation plays a role in the pathogenesis of AMD, EPA may be one of the protective factors in AMD. Supplementation of omega-3 fatty acids, DHA and EPA in particular, has received much interest in association with lowering the risk of AMD. Although DHA can be synthesized from alpha-linolenic acid in the body, the process is ineffective. DHA and EPA can readily be obtained from marine fish oils in the diet. Based on their roles in retinal function and inflammation, dietary modification and supplementation of omega-3 fatty acids have

Many studies have provided evidence for a protective role of omega-3 fatty acids supplementation in AMD risk 91,106-117. The first study evaluating the relationship between dietary fat and AMD was published by Mares-Perlman et al 106. They reported that high intake of saturated fat and cholesterol was associated with increased risk for early AMD. Later, a prospective follow-up study of participants in the Nurses' Health Study and the Health Professionals Follow-up Study showed that total fat intake was positively associated with increased risk of AMD 108. Yet, a cross-sectional study involving participants in the Third National Health and Nutrition Examination Survey found no association between dietary fat and AMD risk. However, this study assessed only one eye per patient, thereby

There are further investigations into the association of omega-3 fatty acids with AMD risk. As dietary omega-3 fatty acids are obtained from marine fish oils, fish intake was also investigated. Earlier study on fish intake was performed in the Blue Mountain Eye Study population. In this cross-sectional, population based study, Smith et al showed that a higher

compared with 2/140 in the placebo group 24,87.

**3.6 Omega-3 Long chain polyunsaturated fatty acids** 

relationship between zinc intake and incidence of early AMD 23. In fact, an early study by the Eye Diseases Case-Control Study Group reported no association between serum zinc levels and risk of neovascular AMD 40. In a 2-year, double-masked, randomized, placebocontrolled study, Stur *et al* reported that oral zinc substitution has no short-term effect in patients who have an exudative form of AMD in one eye 87. Unfortunately, this study was prematurely terminated because of no beneficial effects found in first 40 patients at 24 months. In addition, two large prospective studies involving 66,572 women and 37,636 men do not support a lowered AMD risk associated with higher zinc intake 88. The Blue Mountains Eye Study Group reported no significant association between baseline zinc intake from diet or supplements and the 5-year incidence of early Age-related maculopathy44.

A systematic review and meta-analysis involving four prospective cohort studies 23,42,44,88reported that a pooled odds ratio of zinc for early AMD was 0.91 (95% CI 0.74 to 1.11). Another meta-analysis reported that zinc supplementation can slow down AMD progression (adjusted odds ratio = 0.77, 95% CI 0.62 to 0.96) 89.

Although the evidence is conflicting, recent studies support a protective role of zinc in AMD progression. The AREDS study indicated that the beneficial effect of zinc supplementation was of a similar order to that of vitamin supplementation. Despite the 5-year findings by The Blue Mountains Eye Study Group 44, later studies by the same group published the 10 year data in which individuals with total zinc intake in the highest decile are less likely to develop early or any AMD 45.

Zinc intake and the genetic risk of AMD has also been assessed. In the AREDS population, the single nucleotide polymorphism in the *CFH* (Y402H, rs1061170) and *LOC387715/ARMS2* (A69S, rs10490924) genes of 876 participants who were considered at high risk was genotyped 90. The findings suggest that there is an interaction between *CFH* genotype and treatment with antioxidant plus zinc when compared with placebo. Moreover, a recent study involving 2,167 individuals from the population-based Rotterdam Study at genetic risk of AMD assessed their dietary intake at baseline using a semi-quantitative food frequency questionnaire and determined the genetic variants using TaqMan assay 91. In this nested case-control study, it was observed that there is a significant possibility of biological interaction between CFH Y402H and zinc as well as between LOC387715 A69S and zinc (p < 0.05). Moreover, individuals with homozygous CFH Y402H with dietary intake of zinc in the highest tertile reduced their hazard ratio of early AMD from 2.25 to 1.27.

Again, one has to be cautious about the risks of high dose supplementary intake of zinc. In the AREDS study, more people in the zinc group reported difficulty in swallowing the tablets (17.8% vs. 15.3%, p < 0.04) 19. Circulatory adverse experiences were also more frequently reported in individuals receiving zinc. Hospitalizations due to genitourinary problems as well as mild or moderate symptoms are also more frequent in these participants. In fact, it was found that there is a significant increase in hospital admissions for urinary complications in patients with high zinc supplementation (11.1% vs 7.6%, p = 0.0003) 92. The risk was greatest in male patients (RR 1.26, 95% CI 1.07-1.50, p = 0.008). Significant increase in urinary tract infections was also found (p = 0.004), especially in females. Another problem was gastrointestinal symptoms. Of 286 participants, 5/146 zinc-

relationship between zinc intake and incidence of early AMD 23. In fact, an early study by the Eye Diseases Case-Control Study Group reported no association between serum zinc levels and risk of neovascular AMD 40. In a 2-year, double-masked, randomized, placebocontrolled study, Stur *et al* reported that oral zinc substitution has no short-term effect in patients who have an exudative form of AMD in one eye 87. Unfortunately, this study was prematurely terminated because of no beneficial effects found in first 40 patients at 24 months. In addition, two large prospective studies involving 66,572 women and 37,636 men do not support a lowered AMD risk associated with higher zinc intake 88. The Blue Mountains Eye Study Group reported no significant association between baseline zinc intake from diet or supplements and the 5-year incidence of early Age-related

A systematic review and meta-analysis involving four prospective cohort studies 23,42,44,88reported that a pooled odds ratio of zinc for early AMD was 0.91 (95% CI 0.74 to 1.11). Another meta-analysis reported that zinc supplementation can slow down AMD

Although the evidence is conflicting, recent studies support a protective role of zinc in AMD progression. The AREDS study indicated that the beneficial effect of zinc supplementation was of a similar order to that of vitamin supplementation. Despite the 5-year findings by The Blue Mountains Eye Study Group 44, later studies by the same group published the 10 year data in which individuals with total zinc intake in the highest decile are less likely to

Zinc intake and the genetic risk of AMD has also been assessed. In the AREDS population, the single nucleotide polymorphism in the *CFH* (Y402H, rs1061170) and *LOC387715/ARMS2* (A69S, rs10490924) genes of 876 participants who were considered at high risk was genotyped 90. The findings suggest that there is an interaction between *CFH* genotype and treatment with antioxidant plus zinc when compared with placebo. Moreover, a recent study involving 2,167 individuals from the population-based Rotterdam Study at genetic risk of AMD assessed their dietary intake at baseline using a semi-quantitative food frequency questionnaire and determined the genetic variants using TaqMan assay 91. In this nested case-control study, it was observed that there is a significant possibility of biological interaction between CFH Y402H and zinc as well as between LOC387715 A69S and zinc (p < 0.05). Moreover, individuals with homozygous CFH Y402H with dietary intake of zinc in

Again, one has to be cautious about the risks of high dose supplementary intake of zinc. In the AREDS study, more people in the zinc group reported difficulty in swallowing the tablets (17.8% vs. 15.3%, p < 0.04) 19. Circulatory adverse experiences were also more frequently reported in individuals receiving zinc. Hospitalizations due to genitourinary problems as well as mild or moderate symptoms are also more frequent in these participants. In fact, it was found that there is a significant increase in hospital admissions for urinary complications in patients with high zinc supplementation (11.1% vs 7.6%, p = 0.0003) 92. The risk was greatest in male patients (RR 1.26, 95% CI 1.07-1.50, p = 0.008). Significant increase in urinary tract infections was also found (p = 0.004), especially in females. Another problem was gastrointestinal symptoms. Of 286 participants, 5/146 zinc-

the highest tertile reduced their hazard ratio of early AMD from 2.25 to 1.27.

progression (adjusted odds ratio = 0.77, 95% CI 0.62 to 0.96) 89.

maculopathy44.

develop early or any AMD 45.

treated participants withdrew from the studies due to gastrointestinal symptoms when compared with 2/140 in the placebo group 24,87.

#### **3.6 Omega-3 Long chain polyunsaturated fatty acids**

The retina contains abundant fatty acids, about 30% of which are polyunsaturated fatty acids 93. Polyunsaturated fatty acids are classified into 2 groups: ω-3 and ω-6 depending on the position of the first double bond from the methyl end of the molecule. Docosahexaenoic acid (DHA), an omega-3 fatty acid, is highly enriched in the retina, particularly in the disc membrane of photoreceptor outer segments 94. DHA is the major polyunsaturated fatty acid in cerebral gray matter as well. Yet, the specific role of DHA in the eye is not clear. DHA has been shown to be important for photoreceptor survival 95-98. DHA may have a role in modulating G protein-coupled signaling pathways that are involved in visual transduction 99. DHA may also affect rhodopsin function during photoreception by influencing the membrane's biophysical properties 100,101. In rhesus monkeys, dietary depletion of alphalinolenic acid, a dietary precursor of DHA, resulted in undetectable plasma DHA level and more importantly, abnormal retinogram and visual impairment 102,103. Nonetheless, DHA supplementation is effective in improving retinal function in a patient with autosomal dominant Stargardt-like retinal dystrophy 104. The importance of DHA in retinal function may suggest a possible beneficial role of DHA in retinal disease such as AMD.

Another omega-3 fatty acid, eicosapentaenoic acid (EPA), is the precursor of eicosanoids in the body. It can act as a competitive inhibitor of arachidonic acid conversion to proinflammatory eicosanoids prostaglandin E(2) and leukotriene B(4) 105. As inflammation plays a role in the pathogenesis of AMD, EPA may be one of the protective factors in AMD.

Supplementation of omega-3 fatty acids, DHA and EPA in particular, has received much interest in association with lowering the risk of AMD. Although DHA can be synthesized from alpha-linolenic acid in the body, the process is ineffective. DHA and EPA can readily be obtained from marine fish oils in the diet. Based on their roles in retinal function and inflammation, dietary modification and supplementation of omega-3 fatty acids have become attractive alternatives in lowering the risk of AMD.

Many studies have provided evidence for a protective role of omega-3 fatty acids supplementation in AMD risk 91,106-117. The first study evaluating the relationship between dietary fat and AMD was published by Mares-Perlman et al 106. They reported that high intake of saturated fat and cholesterol was associated with increased risk for early AMD. Later, a prospective follow-up study of participants in the Nurses' Health Study and the Health Professionals Follow-up Study showed that total fat intake was positively associated with increased risk of AMD 108. Yet, a cross-sectional study involving participants in the Third National Health and Nutrition Examination Survey found no association between dietary fat and AMD risk. However, this study assessed only one eye per patient, thereby may have decreased the observed AMD prevalence.

There are further investigations into the association of omega-3 fatty acids with AMD risk. As dietary omega-3 fatty acids are obtained from marine fish oils, fish intake was also investigated. Earlier study on fish intake was performed in the Blue Mountain Eye Study population. In this cross-sectional, population based study, Smith et al showed that a higher

Nutritional Supplement Use and Age-Related Macular Degeneration 201

supported the protective role of omega-3 fatty acids supplementation 118. It was reported that dietary intake of omega-3 fatty acids was associated with reduced risk of late AMD while fish consumption (at least twice a week) was associated with reduced risk of both early and late AMD. However, the authors also cautioned that due to insufficient evidence, few prospective studies and no randomized clinical trials, recommendation for a routine omega-3 fatty acids supplementation and fish consumption for AMD prevention is not supported. A similar conclusion was also reached in another systematic review 119. Hopefully, more definite answers on the protective role of omega-3 fatty acids will be

B vitamins are a group of water-soluble compounds that are important in cell metabolism. The members of interest in AMD studies are folic acid, vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin) because of their ability to reduce homocysteine levels in intervention studies 120. Homocysteine is an amino acid formed during the metabolism of methionine. It can either be recycled back into methionine or converted into cysteine with the help of B-

Serum level of homocysteine has been implicated in increasing the risk of AMD. Recent cross-sectional 121-123and case-control studies 124-128 showed that there may be a direct association between homocysteine level in the blood and AMD. Hyperhomocysteinemia (plasma homocysteine > 15μmol/L) can also induce vascular endothelial dysfunction 129-131. It was therefore proposed that lowering blood homocysteine levels with folic acid, vitamin

In the Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS), 5,442 female health professionals participated in this randomized, double-masked, placebo controlled trial 132. Christen *et al* reported that daily supplementation with folic acid/B6/B12 reduce the risk of AMD in this large cohort of females after an average of 7.3 years of treatment and follow-up. Yet, disease report in this study was done by self-report questionnaires or medical records while no ophthalmic examinations were performed. More evidence and further research in other groups are needed despite the interesting association between folic

Diets rich in fruits, nuts, and vegetables have long been considered to be an excellent source of antioxidants. There has been growing interest on berry extracts due to their high antioxidant properties. Among the berries, blueberries have been of specific interest because of their high antioxidant capacity (in some cases as high as 40−50 μmol Trolox equivalents/g) 133. Indeed, of all the fresh fruits and vegetables tested to date, data indicate that blueberries have the highest antioxidant capacity, as estimated using the average oxygen radical absorbance capacity (ORAC) values 133-135. Polyphenols in blueberries, specifically the anthocyanins that give the fruit its blue color, are the major contributors to

B6 and vitamin B12 supplementation may help to reduce the risk of AMD.

acid/B6/B12 supplementation and AMD prevention.

provided by the ongoing AREDS2 randomized, multi-center trial.

**3.7 B vitamins** 

vitamins.

**3.8 Berry extracts** 

antioxidant activity 133.

fish consumption was associated with decreased odds of late AMD 107. After 5 years of follow-up Chua *et al* reported that fish consumption at least once a week was protective against early AMD, whereas fish consumption at least 3 times per week could reduce the incidence of late AMD 110. After 10 years of follow up in the same cohort, Tan *et al* suggested that a regular weekly serving of fish was associated with a reduced risk of early AMD 116. Interestingly, it was also noted that fish consumption of more than one serving per week did not have a significant protective effect in reducing AMD risk in this cohort, suggesting a threshold effect. These findings are supported by other studies as well. Seddon *et al* in a multicenter eye disease case-control study reported that higher intake of omega-3 fatty acids and fish was associated with a lower risk for AMD among individuals with low linoleic acid intake 109. More evidence on the protective role of omega-3 fatty acid came from a recent US Twin Study of Age-Related Macular Degeneration. This study investigated the association between dietary fat intake and fish consumption and risks of AMD in 681 twins 111 and found that both omega-3 and fish intake reduced the risk of AMD.

Oily fish rich in omega-3 fatty acids are also found to be beneficial in two European studies. The population-based POLANUT study from Southern France found that fatty fish intake was protective against AMD when comparing more than once a month and less than once a month and after multvariate adjustment 112. Interestingly, total and white fish intake has no significant association with AMD risk. Another population-based study, EUREYE, showed that oily fish intake (at least once per week versus less than once per week) was associated with significant reduction of risk for neovascular AMD 114. Similar findings were also observed for either DHA or EPA intake.

Among the AREDS participants, a prospective cohort of individuals with neovascular AMD and central geographic atrophy was also analyzed for the relationship of omega-3 fatty acids and AMD. It was observed that dietary total omega-3 fatty acids or DHA intake was inversely associated with neovascular AMD 113. Similar findings were also observed with fish consumption. Further studies showed that dietary omega-3 fatty acids intake is associated with a decreased risk of progression from bilateral drusen to central geographic atrophy 115.

In addition, the association between omega-3 fatty acids and genetic risk of AMD is investigated. In the Blue Mountains Study group, 1881 participants were genotyped for complement factor H (CFH) genetic variants 117. Wang *et al* reported that AMD risk increased with each additional C allele. Also, weekly compared with less than weekly consumption of fish was associated with reduced late AMD risk in participants with the CC genotype but not the CT or TT genotypes. This study provided evidence that weekly consumption of fish is protective on the development of late AMD, but not early AMD, among individuals with genetic susceptibility to AMD due to the Y402H variant. On the other hand, the dietary intake of 2167 individuals was assessed at baseline in a recent population-based Rotterdam study 91. Ho *et al* reported a possible interaction between EPA/DHA and either *CFH* Y402H or *LOC387715* A69S. The authors also suggested that high dietary intake of omega-3 fatty acids may reduce the risk of early AMD in those who are at high genetic risk.

Taken together, much data suggests that dietary omega-3 fatty acids intake and fish consumption are protective against AMD. Results from a recent meta-analysis also supported the protective role of omega-3 fatty acids supplementation 118. It was reported that dietary intake of omega-3 fatty acids was associated with reduced risk of late AMD while fish consumption (at least twice a week) was associated with reduced risk of both early and late AMD. However, the authors also cautioned that due to insufficient evidence, few prospective studies and no randomized clinical trials, recommendation for a routine omega-3 fatty acids supplementation and fish consumption for AMD prevention is not supported. A similar conclusion was also reached in another systematic review 119. Hopefully, more definite answers on the protective role of omega-3 fatty acids will be provided by the ongoing AREDS2 randomized, multi-center trial.

#### **3.7 B vitamins**

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

fish consumption was associated with decreased odds of late AMD 107. After 5 years of follow-up Chua *et al* reported that fish consumption at least once a week was protective against early AMD, whereas fish consumption at least 3 times per week could reduce the incidence of late AMD 110. After 10 years of follow up in the same cohort, Tan *et al* suggested that a regular weekly serving of fish was associated with a reduced risk of early AMD 116. Interestingly, it was also noted that fish consumption of more than one serving per week did not have a significant protective effect in reducing AMD risk in this cohort, suggesting a threshold effect. These findings are supported by other studies as well. Seddon *et al* in a multicenter eye disease case-control study reported that higher intake of omega-3 fatty acids and fish was associated with a lower risk for AMD among individuals with low linoleic acid intake 109. More evidence on the protective role of omega-3 fatty acid came from a recent US Twin Study of Age-Related Macular Degeneration. This study investigated the association between dietary fat intake and fish consumption and risks of AMD in 681 twins 111 and

Oily fish rich in omega-3 fatty acids are also found to be beneficial in two European studies. The population-based POLANUT study from Southern France found that fatty fish intake was protective against AMD when comparing more than once a month and less than once a month and after multvariate adjustment 112. Interestingly, total and white fish intake has no significant association with AMD risk. Another population-based study, EUREYE, showed that oily fish intake (at least once per week versus less than once per week) was associated with significant reduction of risk for neovascular AMD 114. Similar findings were also

Among the AREDS participants, a prospective cohort of individuals with neovascular AMD and central geographic atrophy was also analyzed for the relationship of omega-3 fatty acids and AMD. It was observed that dietary total omega-3 fatty acids or DHA intake was inversely associated with neovascular AMD 113. Similar findings were also observed with fish consumption. Further studies showed that dietary omega-3 fatty acids intake is associated with a decreased risk of progression from bilateral drusen to central geographic

In addition, the association between omega-3 fatty acids and genetic risk of AMD is investigated. In the Blue Mountains Study group, 1881 participants were genotyped for complement factor H (CFH) genetic variants 117. Wang *et al* reported that AMD risk increased with each additional C allele. Also, weekly compared with less than weekly consumption of fish was associated with reduced late AMD risk in participants with the CC genotype but not the CT or TT genotypes. This study provided evidence that weekly consumption of fish is protective on the development of late AMD, but not early AMD, among individuals with genetic susceptibility to AMD due to the Y402H variant. On the other hand, the dietary intake of 2167 individuals was assessed at baseline in a recent population-based Rotterdam study 91. Ho *et al* reported a possible interaction between EPA/DHA and either *CFH* Y402H or *LOC387715* A69S. The authors also suggested that high dietary intake of omega-3 fatty acids may reduce the risk of early AMD in those who

Taken together, much data suggests that dietary omega-3 fatty acids intake and fish consumption are protective against AMD. Results from a recent meta-analysis also

found that both omega-3 and fish intake reduced the risk of AMD.

observed for either DHA or EPA intake.

atrophy 115.

are at high genetic risk.

B vitamins are a group of water-soluble compounds that are important in cell metabolism. The members of interest in AMD studies are folic acid, vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin) because of their ability to reduce homocysteine levels in intervention studies 120. Homocysteine is an amino acid formed during the metabolism of methionine. It can either be recycled back into methionine or converted into cysteine with the help of Bvitamins.

Serum level of homocysteine has been implicated in increasing the risk of AMD. Recent cross-sectional 121-123and case-control studies 124-128 showed that there may be a direct association between homocysteine level in the blood and AMD. Hyperhomocysteinemia (plasma homocysteine > 15μmol/L) can also induce vascular endothelial dysfunction 129-131. It was therefore proposed that lowering blood homocysteine levels with folic acid, vitamin B6 and vitamin B12 supplementation may help to reduce the risk of AMD.

In the Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS), 5,442 female health professionals participated in this randomized, double-masked, placebo controlled trial 132. Christen *et al* reported that daily supplementation with folic acid/B6/B12 reduce the risk of AMD in this large cohort of females after an average of 7.3 years of treatment and follow-up. Yet, disease report in this study was done by self-report questionnaires or medical records while no ophthalmic examinations were performed. More evidence and further research in other groups are needed despite the interesting association between folic acid/B6/B12 supplementation and AMD prevention.

#### **3.8 Berry extracts**

Diets rich in fruits, nuts, and vegetables have long been considered to be an excellent source of antioxidants. There has been growing interest on berry extracts due to their high antioxidant properties. Among the berries, blueberries have been of specific interest because of their high antioxidant capacity (in some cases as high as 40−50 μmol Trolox equivalents/g) 133. Indeed, of all the fresh fruits and vegetables tested to date, data indicate that blueberries have the highest antioxidant capacity, as estimated using the average oxygen radical absorbance capacity (ORAC) values 133-135. Polyphenols in blueberries, specifically the anthocyanins that give the fruit its blue color, are the major contributors to antioxidant activity 133.

Nutritional Supplement Use and Age-Related Macular Degeneration 203

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Anthocyanin is a water-soluble pigment present in all plants and is richly concentrated in berries. It is a powerful antioxidant *in vitro* 136. It can absorb blue-green light and protects the cells from light stress in plant studies 137. In laboratory studies, anthocyanin may protect the eyes from degenerative diseases such as AMD 138-140. Yet, the evidence for the potential health effects of anthocyanin is mostly laboratory-based 141.

Another berry that recently received lots of interest is the fruit of *Lycium barbaurm*, also called wolfberry or Gouqizi, a commonly used herb in Chinese Traditional Medicine. It is also taken as food in Asian countries. It is well known for improving eye sight. Increasing lines of evidence showed that the polysaccharides in *Lycium barbaurm* can exhibit anti-aging 142 and anti-oxidative effects 143. Other properties such as anti-tumor effects, cytoprotection, neuromodulation, and immune modulations have also been suggested 142,144. Unfortunately, most evidence for its beneficial effects is limited to the laboratory level.

At this moment, there are no legal requirements for quality control in the preparation of these extracts. It is not obligatory to disclose the content and the production method. Moreover, the dosage and frequency are unclear while potential toxicity and long-term side effects remain to be investigated. A lot of investigation is needed before the potential of berry extracts in prevention of AMD can be hinted. Currently, berry extracts should not be recommended 27.

#### **4. Future directions**

Observational studies have shown beneficial effects from dietary supplementation of lutein and zeaxanthin as well as omega-3 fatty acids in the development of AMD. They are currently tested in AREDS2, the multi-centered randomized clinical trial launched by the National Eye Institute in 2006. The association of oral formulations containing lutein and zeaxanthin, and/or DHA and EPA, with the progression of AMD is being assessed. In AREDS2 participants will be followed for 5 years. Hopefully, data will be available by the end of 2012. Similarly, the ongoing CARMA study will also provide invaluable data on the protective effects of lutein and zeaxanthin in combination with antioxidants (vitamin C, vitamin E and zinc) with the exclusion of DHA and EPA.

#### **5. Conclusions**

To date a large body of evidence has supported a protective role of nutritional supplements in the development and progression of AMD. In particular, strongest evidence is present for the protective effect of lutein, zeaxanthin, DHA, and EPA. On the other hand, beta-carotene and vitamin E may have detrimental effects. While awaiting a further proof of the effects of lutein, zeaxanthin, DHA, and EPA, the AREDS formulation remains the best recommendation so far, although not without risk and maybe only for high-risk individuals. One concern for the AREDS formulation is the higher risk of lung cancer in smokers with daily beta-carotene supplementation. Therefore, in offering nutritional supplements to patients, physicians should consider on a case-by-case basis and fully explain the potential side effects from a long-term regular intake. It is also important to remind the patients that even with the AREDS formulation, AMD can still occur. It is equally important to teach the patients self-monitoring methods such as usage of the Amsler grid. Regular fundal examinations by ophthalmologists should also be strongly encouraged.

#### **6. Reference**

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

Anthocyanin is a water-soluble pigment present in all plants and is richly concentrated in berries. It is a powerful antioxidant *in vitro* 136. It can absorb blue-green light and protects the cells from light stress in plant studies 137. In laboratory studies, anthocyanin may protect the eyes from degenerative diseases such as AMD 138-140. Yet, the evidence for the potential

Another berry that recently received lots of interest is the fruit of *Lycium barbaurm*, also called wolfberry or Gouqizi, a commonly used herb in Chinese Traditional Medicine. It is also taken as food in Asian countries. It is well known for improving eye sight. Increasing lines of evidence showed that the polysaccharides in *Lycium barbaurm* can exhibit anti-aging 142 and anti-oxidative effects 143. Other properties such as anti-tumor effects, cytoprotection, neuromodulation, and immune modulations have also been suggested 142,144. Unfortunately,

At this moment, there are no legal requirements for quality control in the preparation of these extracts. It is not obligatory to disclose the content and the production method. Moreover, the dosage and frequency are unclear while potential toxicity and long-term side effects remain to be investigated. A lot of investigation is needed before the potential of berry extracts in prevention of AMD can be hinted. Currently, berry extracts should not be

Observational studies have shown beneficial effects from dietary supplementation of lutein and zeaxanthin as well as omega-3 fatty acids in the development of AMD. They are currently tested in AREDS2, the multi-centered randomized clinical trial launched by the National Eye Institute in 2006. The association of oral formulations containing lutein and zeaxanthin, and/or DHA and EPA, with the progression of AMD is being assessed. In AREDS2 participants will be followed for 5 years. Hopefully, data will be available by the end of 2012. Similarly, the ongoing CARMA study will also provide invaluable data on the protective effects of lutein and zeaxanthin in combination with antioxidants (vitamin C,

To date a large body of evidence has supported a protective role of nutritional supplements in the development and progression of AMD. In particular, strongest evidence is present for the protective effect of lutein, zeaxanthin, DHA, and EPA. On the other hand, beta-carotene and vitamin E may have detrimental effects. While awaiting a further proof of the effects of lutein, zeaxanthin, DHA, and EPA, the AREDS formulation remains the best recommendation so far, although not without risk and maybe only for high-risk individuals. One concern for the AREDS formulation is the higher risk of lung cancer in smokers with daily beta-carotene supplementation. Therefore, in offering nutritional supplements to patients, physicians should consider on a case-by-case basis and fully explain the potential side effects from a long-term regular intake. It is also important to remind the patients that even with the AREDS formulation, AMD can still occur. It is equally important to teach the patients self-monitoring methods such as usage of the Amsler grid. Regular fundal

health effects of anthocyanin is mostly laboratory-based 141.

vitamin E and zinc) with the exclusion of DHA and EPA.

examinations by ophthalmologists should also be strongly encouraged.

recommended 27.

**5. Conclusions** 

**4. Future directions** 

most evidence for its beneficial effects is limited to the laboratory level.


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**11** 

*Canada* 

**Two-Photon Excitation Photodynamic** 

Ira Probodh and David Thomas Cramb *Department of Chemistry, University of Calgary* 

**Therapy: Working Toward a New Treatment** 

**for Wet Age-Related Macular Degeneration** 

Photodynamic therapy (PDT) exploits the cytotoxic effects of light-activated compounds to achieve spatially selective tissue eradication. It is used in treating a wide range of tumors (Lou et al., 2003), localized infections (Hamblin & Hasan, 2004), and diseases like the wet form of age-related macular degeneration (Bressler & Bressler, 2000). The treatment involves application of a non-toxic photosensitizer that is preferentially taken up by the target cells/tissue. Optical excitation of the photosensitizer produces reactive oxygen species that cause localised, apoptotic cell death. Herein we review the application of a new modality – two-photon excitation-PDT (TPE-PDT) - to the treatment of wet age-related macular degeneration (wet-AMD). We show that the application of TPE-PDT, in conjunction with newly developed photosensitizers, has the potential to greatly improve therapy of wet-

Wet-AMD is characterised by generation of blood vessels in the normally avascular retinal macula. The newly formed blood vessels leak fluid and/or blood under the macula, leading to rapid vision loss through damage to the photoreceptors (Rattner & Nathans, 2006). PDT, using single photon activation of the photosensitizer Verteporfin (trade name Visudyne), has been used for the treatment of wet-AMD since 2000 (Bressler & Bressler, 2000). In the clinic, verteporfin is first administered to patients through systemic injections (Soubrane & Bressler, 2001), and the photosensitizer accumulates in areas of high cellular reproduction like the neovasculature in the retinal tissue. Photo-irradiation of the photosensitizer leads to a localised, Type II photoreaction associated with singlet oxygen generation (Schmidt-Erfurth & Hasan, 2000). The photosensitizer absorbs a photon and is promoted to the excited singlet state that converts to an excited triplet state through intersystem crossing. An energy transfer between the triplet excited state of photosensitizer and naturally occurring triplet oxygen then produces reactive singlet oxygen. While Type I reactions involving radicals are also possible, PDT is generally accepted as occurring predominantly through the singlet

**1.1 Why two-photon photodynamic therapy (TPE-PDT)?** 

**1.1.1 Photodynamic therapy (PDT)** 

oxygen mechanism.

**1. Introduction** 

AMD.


### **Two-Photon Excitation Photodynamic Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration**

Ira Probodh and David Thomas Cramb *Department of Chemistry, University of Calgary Canada* 

#### **1. Introduction**

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

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62.

against aging-associated diseases. What do we know so far? *Cell Mol Neurobiol*

Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury. *PLoS One*

they be used in aging-associated neurodegenerative diseases? *Ageing Res Rev* 9:354-

Relationship of dietary fat to age-related maculopathy in the Third National Health

and risk of age-related macular degeneration in the age-related eye disease study.

Photodynamic therapy (PDT) exploits the cytotoxic effects of light-activated compounds to achieve spatially selective tissue eradication. It is used in treating a wide range of tumors (Lou et al., 2003), localized infections (Hamblin & Hasan, 2004), and diseases like the wet form of age-related macular degeneration (Bressler & Bressler, 2000). The treatment involves application of a non-toxic photosensitizer that is preferentially taken up by the target cells/tissue. Optical excitation of the photosensitizer produces reactive oxygen species that cause localised, apoptotic cell death. Herein we review the application of a new modality – two-photon excitation-PDT (TPE-PDT) - to the treatment of wet age-related macular degeneration (wet-AMD). We show that the application of TPE-PDT, in conjunction with newly developed photosensitizers, has the potential to greatly improve therapy of wet-AMD.

#### **1.1 Why two-photon photodynamic therapy (TPE-PDT)?**

#### **1.1.1 Photodynamic therapy (PDT)**

Wet-AMD is characterised by generation of blood vessels in the normally avascular retinal macula. The newly formed blood vessels leak fluid and/or blood under the macula, leading to rapid vision loss through damage to the photoreceptors (Rattner & Nathans, 2006). PDT, using single photon activation of the photosensitizer Verteporfin (trade name Visudyne), has been used for the treatment of wet-AMD since 2000 (Bressler & Bressler, 2000). In the clinic, verteporfin is first administered to patients through systemic injections (Soubrane & Bressler, 2001), and the photosensitizer accumulates in areas of high cellular reproduction like the neovasculature in the retinal tissue. Photo-irradiation of the photosensitizer leads to a localised, Type II photoreaction associated with singlet oxygen generation (Schmidt-Erfurth & Hasan, 2000). The photosensitizer absorbs a photon and is promoted to the excited singlet state that converts to an excited triplet state through intersystem crossing. An energy transfer between the triplet excited state of photosensitizer and naturally occurring triplet oxygen then produces reactive singlet oxygen. While Type I reactions involving radicals are also possible, PDT is generally accepted as occurring predominantly through the singlet oxygen mechanism.

Two-Photon Excitation Photodynamic

One-photon

excitation (Goyan & Cramb, 2000; Samkoe et al., 2006).

Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration 215

molecule from the virtual intermediate state to the singlet excited state. The probability of this event is very small, and it is proportional to the square of the light intensity. Two-photon absorption, therefore, occurs only at the focal plane of a tightly focused laser beam (Goyan et al., 2001; Oheim et al., 2006). It should be noted that the excited states achieved by one and two-photon absorption are identical. The photophysical and photochemical properties of the photosensitizer are thus, unaffected by the mode of

excitation Two-photon

excitation

Fig. 2. In one photon excitation, photosensitizer activation occurs throughout the path of the laser beam, but in two-photon excitation, activation occurs only at the focus of the laser beam (red oval in right panel). The localised excitation in TPE-PDT is likely to cause less

Typically, excitation volumes of a few femtoliters can be achieved with two-photon absorption. This extremely confined excitation volume allows for high spatial selectivity. Use of a TPE treatment modality therefore, has the potential to selectively excite photosensitizer in the neovasculature, leaving the surrounding tissue unaffected (see Figure 2). In the following sections we review the experiments that demonstrate TPE-PDT *in vitro* and vessel occlusion *in vivo* with clinically approved and novel photosensitizers, and discuss

While *in vivo* experiments in animal models are essential to test and demonstrate the efficacy of drugs, it is very useful to quickly pre-screen drugs in cellular models before expending

collateral damage. [Adapted from PhD thesis of K. S. Samkoe (Samkoe, 2007)]

**2. TPE-PDT in vitro – Testing photosensitizers in cell-lines** 

the implications of its therapeutic use.

The PDT-induced vessel occlusion, *in vivo*, is generally attributed to singlet oxygen mediated direct vascular damage of blood vessel endothelium. This initiates a cascade of responses which include platelet aggregation, leukocyte adhesion, vascular permeabilization and vasoconstriction (Krammer, 2001). These, in turn, are expected to cause vascular occlusion.

The short lifetime of singlet oxygen (3.5µs in aqueous environment (Pervaiz, 2001)) ensures that the area affected by it is spatially confined to a small volume. It is estimated that singlet oxygen can diffuse to a distance of around 100nm or less (Skovsen et al., 2005) *in vivo*. PDT, thus, offers a relatively selective and non-invasive method to occlude the abnormal vascularization characteristic of wet-AMD. The stages in PDT for treatment of wet-AMD are diagrammed in Figure 1.

Fig. 1. Visual representation of Verteporfin photodynamic therapy: injected verteporfin accumulates in the retinal neovasculature, where it is activated by illumination with a 680nm laser beam. Laser activation leads to singlet oxygen production that in turn leads to vessel occlusion. [Adapted from PhD thesis of K. S. Samkoe (Samkoe, 2007)]

Currently, clinical PDT treatment involves excitation of Verteporfin with 689nm laser light that excites via a one-photon absorption peak in the so-called Q-band of the photosensitizer. The disadvantage of this treatment regime is that one-photon excitation can damage the over- and underlying tissues adjacent to the treated area, through excitation of photosensitizer present there (Reinke et al., 1999). This deleterious side-effect can be reduced by using two-photon excitation of photosensitizer.

#### **1.1.2 Two-photon excitation PDT (TPE-PDT)**

Two-photon excitation (TPE) of fluorophores is extensively utilized in confocal microscopy (Oheim et al., 2006; So et al., 2000). Because two-photon absorption cross-sections are very small, excitation requires high fluxes of light that can be achieved by using a tightly focused femtosecond laser beam as the light source. In TPE, a molecule is excited by simultaneous absorption of two photons of half the energy, or twice the wavelength, of one photon excitation. The first photon excites the molecule from its ground state to a virtual intermediate excited state. A second photon, simultaneously absorbed, promotes the

The PDT-induced vessel occlusion, *in vivo*, is generally attributed to singlet oxygen mediated direct vascular damage of blood vessel endothelium. This initiates a cascade of responses which include platelet aggregation, leukocyte adhesion, vascular permeabilization and vasoconstriction (Krammer, 2001). These, in turn, are expected to

The short lifetime of singlet oxygen (3.5µs in aqueous environment (Pervaiz, 2001)) ensures that the area affected by it is spatially confined to a small volume. It is estimated that singlet oxygen can diffuse to a distance of around 100nm or less (Skovsen et al., 2005) *in vivo*. PDT, thus, offers a relatively selective and non-invasive method to occlude the abnormal vascularization characteristic of wet-AMD. The stages in PDT for treatment of wet-AMD are

Fig. 1. Visual representation of Verteporfin photodynamic therapy: injected verteporfin accumulates in the retinal neovasculature, where it is activated by illumination with a 680nm laser beam. Laser activation leads to singlet oxygen production that in turn leads to

Verteporfin + O2 + *hν* 1O2 Vessel closure

Currently, clinical PDT treatment involves excitation of Verteporfin with 689nm laser light that excites via a one-photon absorption peak in the so-called Q-band of the photosensitizer. The disadvantage of this treatment regime is that one-photon excitation can damage the over- and underlying tissues adjacent to the treated area, through excitation of photosensitizer present there (Reinke et al., 1999). This deleterious side-effect can be

Two-photon excitation (TPE) of fluorophores is extensively utilized in confocal microscopy (Oheim et al., 2006; So et al., 2000). Because two-photon absorption cross-sections are very small, excitation requires high fluxes of light that can be achieved by using a tightly focused femtosecond laser beam as the light source. In TPE, a molecule is excited by simultaneous absorption of two photons of half the energy, or twice the wavelength, of one photon excitation. The first photon excites the molecule from its ground state to a virtual intermediate excited state. A second photon, simultaneously absorbed, promotes the

vessel occlusion. [Adapted from PhD thesis of K. S. Samkoe (Samkoe, 2007)]

reduced by using two-photon excitation of photosensitizer.

**1.1.2 Two-photon excitation PDT (TPE-PDT)** 

cause vascular occlusion.

diagrammed in Figure 1.

molecule from the virtual intermediate state to the singlet excited state. The probability of this event is very small, and it is proportional to the square of the light intensity. Two-photon absorption, therefore, occurs only at the focal plane of a tightly focused laser beam (Goyan et al., 2001; Oheim et al., 2006). It should be noted that the excited states achieved by one and two-photon absorption are identical. The photophysical and photochemical properties of the photosensitizer are thus, unaffected by the mode of excitation (Goyan & Cramb, 2000; Samkoe et al., 2006).

Fig. 2. In one photon excitation, photosensitizer activation occurs throughout the path of the laser beam, but in two-photon excitation, activation occurs only at the focus of the laser beam (red oval in right panel). The localised excitation in TPE-PDT is likely to cause less collateral damage. [Adapted from PhD thesis of K. S. Samkoe (Samkoe, 2007)]

Typically, excitation volumes of a few femtoliters can be achieved with two-photon absorption. This extremely confined excitation volume allows for high spatial selectivity. Use of a TPE treatment modality therefore, has the potential to selectively excite photosensitizer in the neovasculature, leaving the surrounding tissue unaffected (see Figure 2). In the following sections we review the experiments that demonstrate TPE-PDT *in vitro* and vessel occlusion *in vivo* with clinically approved and novel photosensitizers, and discuss the implications of its therapeutic use.

#### **2. TPE-PDT in vitro – Testing photosensitizers in cell-lines**

While *in vivo* experiments in animal models are essential to test and demonstrate the efficacy of drugs, it is very useful to quickly pre-screen drugs in cellular models before expending

Two-Photon Excitation Photodynamic

**3. TPE-PDT in vivo** 

Samkoe (Samkoe, 2007).]

**3.1.1 The CAM as a model for wet-AMD** 

model for the neovasculature occurring in wet-AMD.

 **Chicken embryo** 

the involvement of two-photon processes in PDT.

candidate for future development as a TPE-PDT photosensitizer.

**3.1 Complete occlusion of neovasculature in the chicken CAM** 

Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration 217

non-linear dependence of TPE-PDT on light intensity, providing unambiguous support of

While utilizing clinically approved photosensitizers for TPE-PDT is very attractive, their small two-photon absorption may be limiting in the clinical context. Collins et al (Collins et al., 2008) reported the use of "designer" TPE-PDT drugs – novel porphyrin dimers specifically designed to have high two-photon absorption cross section. The authors tested a series of porphyrin dimers for their PDT-induced cytotoxicity in a cancer cell line and demonstrated the higher TPE-PDT efficiency of dimer 1 (fig. 3) relative to verteporfin. Dimer 1 was then selected for *in vivo* TPE-PDT testing [section 3.2.2] and is a strong

The chicken chorioallantoic membrane (CAM) is a transparent extra-embryonic membrane that grows against the inner wall of the developing egg. It is an external lung and waste exchange system for the embryo, and has a wide range of blood vessel sizes – from a few microns to several hundred microns in diameter (Patten, 1971). This makes it easy to find blood vessels that are similar in size to the neovasculature produced in the human eye during AMD (see Figure 4), and like the latter, the blood vessels are undergoing rapid angiogenesis between days 5 to 9 of gestation (Schlatter et al., 1997). The chicken embryo has a short gestation period and is easy to manipulate. It is as such, an inexpensive and useful

**Human retina** 

Fig. 4. The chicken chorio-allantoic membrane (CAM) exhibits blood vessels similar in size to those in the human eye, making it a good model for the neovasculature in wet-AMD. [Picture of chicken embryo reproduced with permission from Clancy et al., 2010, *Chemical Physics Letters,* 488, 99-111; picture of human retina reproduced from PhD thesis of K. S.

time and effort on laborious animal experiments. Cells can grow faster than animals, are inexpensive to maintain and easier to handle. In the context of PDT, they are particularly useful since cell death post-PDT treatment can be easily quantified.

Fig. 3. The photosensitizers [Porphyrin dimers adapted from (Collins et al., 2008)]

Khurana et al (Khurana et al., 2007) have used endothelial cells to assess the TPE-PDT efficacy of photosensitizers approved for one photon PDT – namely, photofrin and verteporfin. They incubated a confluent monolayer of endothelial cells (YPEN-1) with each photosensitizer and performed TPE-PDT by irradiating the cell layer with the 865nm laser line of a femtosecond Ti:Sapphire laser at various output powers. TPE-PDT effect was quantified by using cell permeability stains to assess cell death post-treatment. The authors achieved TPE-PDT induced cell death with both photosensitizers, but Verteporfin was around seven times more effective, consistent with its higher two-photon cross-section. Using verteporfin and by varying the laser dose, they were also able to demonstrate the non-linear dependence of TPE-PDT on light intensity, providing unambiguous support of the involvement of two-photon processes in PDT.

While utilizing clinically approved photosensitizers for TPE-PDT is very attractive, their small two-photon absorption may be limiting in the clinical context. Collins et al (Collins et al., 2008) reported the use of "designer" TPE-PDT drugs – novel porphyrin dimers specifically designed to have high two-photon absorption cross section. The authors tested a series of porphyrin dimers for their PDT-induced cytotoxicity in a cancer cell line and demonstrated the higher TPE-PDT efficiency of dimer 1 (fig. 3) relative to verteporfin. Dimer 1 was then selected for *in vivo* TPE-PDT testing [section 3.2.2] and is a strong candidate for future development as a TPE-PDT photosensitizer.

### **3. TPE-PDT in vivo**

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

time and effort on laborious animal experiments. Cells can grow faster than animals, are inexpensive to maintain and easier to handle. In the context of PDT, they are particularly

Fig. 3. The photosensitizers [Porphyrin dimers adapted from (Collins et al., 2008)]

Khurana et al (Khurana et al., 2007) have used endothelial cells to assess the TPE-PDT efficacy of photosensitizers approved for one photon PDT – namely, photofrin and verteporfin. They incubated a confluent monolayer of endothelial cells (YPEN-1) with each photosensitizer and performed TPE-PDT by irradiating the cell layer with the 865nm laser line of a femtosecond Ti:Sapphire laser at various output powers. TPE-PDT effect was quantified by using cell permeability stains to assess cell death post-treatment. The authors achieved TPE-PDT induced cell death with both photosensitizers, but Verteporfin was around seven times more effective, consistent with its higher two-photon cross-section. Using verteporfin and by varying the laser dose, they were also able to demonstrate the

useful since cell death post-PDT treatment can be easily quantified.

#### **3.1 Complete occlusion of neovasculature in the chicken CAM**

#### **3.1.1 The CAM as a model for wet-AMD**

The chicken chorioallantoic membrane (CAM) is a transparent extra-embryonic membrane that grows against the inner wall of the developing egg. It is an external lung and waste exchange system for the embryo, and has a wide range of blood vessel sizes – from a few microns to several hundred microns in diameter (Patten, 1971). This makes it easy to find blood vessels that are similar in size to the neovasculature produced in the human eye during AMD (see Figure 4), and like the latter, the blood vessels are undergoing rapid angiogenesis between days 5 to 9 of gestation (Schlatter et al., 1997). The chicken embryo has a short gestation period and is easy to manipulate. It is as such, an inexpensive and useful model for the neovasculature occurring in wet-AMD.

Fig. 4. The chicken chorio-allantoic membrane (CAM) exhibits blood vessels similar in size to those in the human eye, making it a good model for the neovasculature in wet-AMD. [Picture of chicken embryo reproduced with permission from Clancy et al., 2010, *Chemical Physics Letters,* 488, 99-111; picture of human retina reproduced from PhD thesis of K. S. Samkoe (Samkoe, 2007).]

Two-Photon Excitation Photodynamic

*Biomedical Optics*, 14, 064006.]

(fig.6).

Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration 219

38mW (corresponding to a fluence of 1.1x108J/cm2) and treatment time of 5min achieved complete occlusion of these small arteries immediately after the treatment. As expected, increasing the drug dose, the laser power, and/or the laser treatment time increases the efficacy of occlusion, presumably through increased singlet oxygen generation. By increasing the laser power and treatment times, it was also possible to achieve long-term occlusion of blood vessels. A minimum of 45mW laser power and a laser treatment time of 3min were required for long term occlusion of up to 50μm arteries (Khurana et al., 2009)

Fig. 6. Optimising laser dosage to occlude 50µm arteries. A vessel closure rating of 4 equals complete occlusion. [Adapted with permission from Khurana et al., (2009) *Journal of* 

60

180

Treatment

0

300

 time (s)

1

Ve

s

s

el clos

ure

ratin

g

2

3

4

30

40

(mW)

50

power

Laser

Clearly, increasing the laser power and laser treatment times has the potential to improve occlusion efficacy of TPE-PDT and shut down larger feeder vessels. So is there a glass ceiling? The two major limiting factors are the laser's maximum power output which limits the maximum fluence, and the photosensitizer/oxygen concentration in the excitation volume, which limits the singlet oxygen generated during the treatment. In our experiments, it appears to be the latter, with the TPE-PDT efficacy levelling off at laser powers of 120mW for the highest drug dose tried (2 mg per kg of body weight). The option of increasing the treatment times is not preferred as longer treatment times reduce the long-

#### **3.1.2 Experimental set-up for PDT in the chicken embryo**

The following protocol to prepare the embryo was developed in the Cramb group (Samkoe et al., 2007). Chicken eggs are incubated at 37°C and 60% humidity for 9 days prior to the experiment. On day 4.5, the eggs are "windowed" by first draining 3-4ml of albumen from the blunt end using a syringe, and then cutting a small window in the egg-shell. The window is then covered by cellulose tape for the duration of the incubation, and widened before the experiment to facilitate injection.

Fig. 5. TPE-PDT in the chicken CAM. Photosensitizer is injected with the microinjector attached to the custom-built stage and TPE-PDT is carried out by focusing the laser beam on the treated artery through the microscope objective. [Picture reproduced from PhD thesis of Y. Gregoriou (Gregoriou, 2011)]

For the TPE-PDT experiments, the embryo was mounted on an upright fluorescence microscope with a custom designed sample stage (Figure 5). Verteporfin was administered as a vesicle preparation, injected intrarterially or intravenously, using a microinjector attached to the sample stage. 10min after injection, PDT was performed by directing the light (780nm) of a Ti:Sapphire laser (pulse duration ~ 100fsec) into the selected artery. Laser power of the incident light, duration of the laser illumination and the number of laser treatments were varied to achieve optimal PDT treatment. Vessel occlusion was monitored by taking video images of the treated areas before and after PDT treatment. For tracking long term occlusion, embryos were monitored for up to 6hrs after TPE-PDT treatment. For the multiple short laser treatments, each treatment was performed by focusing the laser beam on to the upper wall of the blood vessel for the required time, then moving it to another spot, close by, on the same artery.

#### **3.1.3 Vessel occlusion by TPE-PDT in the chicken embryo**

The first experiments demonstrating TPE-PDT-induced complete occlusion of blood vessels in the CAM occluded up to 15µm diameter arteries (Samkoe et al., 2007). A laser power of

The following protocol to prepare the embryo was developed in the Cramb group (Samkoe et al., 2007). Chicken eggs are incubated at 37°C and 60% humidity for 9 days prior to the experiment. On day 4.5, the eggs are "windowed" by first draining 3-4ml of albumen from the blunt end using a syringe, and then cutting a small window in the egg-shell. The window is then covered by cellulose tape for the duration of the incubation, and widened

Fig. 5. TPE-PDT in the chicken CAM. Photosensitizer is injected with the microinjector attached to the custom-built stage and TPE-PDT is carried out by focusing the laser beam on the treated artery through the microscope objective. [Picture reproduced from PhD thesis of

For the TPE-PDT experiments, the embryo was mounted on an upright fluorescence microscope with a custom designed sample stage (Figure 5). Verteporfin was administered as a vesicle preparation, injected intrarterially or intravenously, using a microinjector attached to the sample stage. 10min after injection, PDT was performed by directing the light (780nm) of a Ti:Sapphire laser (pulse duration ~ 100fsec) into the selected artery. Laser power of the incident light, duration of the laser illumination and the number of laser treatments were varied to achieve optimal PDT treatment. Vessel occlusion was monitored by taking video images of the treated areas before and after PDT treatment. For tracking long term occlusion, embryos were monitored for up to 6hrs after TPE-PDT treatment. For the multiple short laser treatments, each treatment was performed by focusing the laser beam on to the upper wall of the blood vessel for the required time, then moving it to

Custom-built egg holder

The first experiments demonstrating TPE-PDT-induced complete occlusion of blood vessels in the CAM occluded up to 15µm diameter arteries (Samkoe et al., 2007). A laser power of

**3.1.2 Experimental set-up for PDT in the chicken embryo** 

before the experiment to facilitate injection.

Y. Gregoriou (Gregoriou, 2011)]

microinjector

another spot, close by, on the same artery.

**3.1.3 Vessel occlusion by TPE-PDT in the chicken embryo** 

38mW (corresponding to a fluence of 1.1x108J/cm2) and treatment time of 5min achieved complete occlusion of these small arteries immediately after the treatment. As expected, increasing the drug dose, the laser power, and/or the laser treatment time increases the efficacy of occlusion, presumably through increased singlet oxygen generation. By increasing the laser power and treatment times, it was also possible to achieve long-term occlusion of blood vessels. A minimum of 45mW laser power and a laser treatment time of 3min were required for long term occlusion of up to 50μm arteries (Khurana et al., 2009) (fig.6).

Fig. 6. Optimising laser dosage to occlude 50µm arteries. A vessel closure rating of 4 equals complete occlusion. [Adapted with permission from Khurana et al., (2009) *Journal of Biomedical Optics*, 14, 064006.]

Clearly, increasing the laser power and laser treatment times has the potential to improve occlusion efficacy of TPE-PDT and shut down larger feeder vessels. So is there a glass ceiling? The two major limiting factors are the laser's maximum power output which limits the maximum fluence, and the photosensitizer/oxygen concentration in the excitation volume, which limits the singlet oxygen generated during the treatment. In our experiments, it appears to be the latter, with the TPE-PDT efficacy levelling off at laser powers of 120mW for the highest drug dose tried (2 mg per kg of body weight). The option of increasing the treatment times is not preferred as longer treatment times reduce the long-

Two-Photon Excitation Photodynamic

Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration 221

Fig. 8. Blood vessel occlusion after TPE-PDT. 2 mg/kg Verteporfin liposomal solution injected intravenously; 3×30s treatments with 70mW laser achieved almost complete

**3.2.1 The mouse window chamber model for testing vascular occlusion** 

positions of the laser beam during TPE-PDT treatment)

**3.2 Vascular occlusion in the mouse window model** 

occlusion of a 55µm artery immediately after treatment. (The red dots mark the approximate

Khurana et al (Khurana et al., 2009) have investigated TPE-PDT in a murine chamber window model. Surgical placement of a transparent window (1cm diameter) into the dorsal skin of a mouse allows for direct visualization of skin vasculature and administration of PDT treatment under a confocal laser scanning microscope (see Figure 9). This has the advantage of using a more robust animal model that allows for tracking of vascular occlusion on the long term - up to 25hrs. With the chicken CAM model, it is typically possible to track vessel occlusion only up to 6-7hrs after the treatment. The disadvantage of using the mouse model for wet-AMD is that the blood vessels tested are normal healthy vessels, not leaky neovasculature of the type found in wet-AMD and the chicken CAM.

Fig. 9. The murine window chamber model. Nude mouse with surgically implanted

from Khurana et al., (2009) *Journal of Biomedical Optics*, 14, 064006]

window (left), vasculature visible through the window (right) [Reproduced with permission

term viability of the embryo and limit the usefulness of the model in terms of tracking longterm occlusion. Moreover, for patient compliance during TPE-PDT, shorter treatment times are preferred.

Fig. 7. Varying the laser dosage and treatment regimes to occlude large blood vessels shows that multiple short treatments are more useful than long single treatments in vessel occlusion.

Multiple short laser treatments, interspersed with 'dark' periods, could overcome these limitations by exciting a large number of photosensitizers in a short period, and then allowing the blood flow to replenish the ground state photosensitizer and/or oxygen during the 'dark' period. The examples in Fig. 7 and 8 clearly indicate that multiple short laser treatments are more efficacious than single long treatments to achieve long-term occlusion of large vessels. We were able to completely occlude up to 100μm sized arteries using laser power as low as 70mW with 30sx3 laser treatments, whereas a laser power of 120mW and 120s treatment time only succeeded in occluding up to 60μm arteries. Our data suggest that in the context of clinical verteporfin TPE-PDT treatment, it would be useful to investigate multiple short laser treatments to achieve optimal PDT efficiency and shut down large feeder vessels.

Fig. 8. Blood vessel occlusion after TPE-PDT. 2 mg/kg Verteporfin liposomal solution injected intravenously; 3×30s treatments with 70mW laser achieved almost complete occlusion of a 55µm artery immediately after treatment. (The red dots mark the approximate positions of the laser beam during TPE-PDT treatment)

#### **3.2 Vascular occlusion in the mouse window model**

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

term viability of the embryo and limit the usefulness of the model in terms of tracking longterm occlusion. Moreover, for patient compliance during TPE-PDT, shorter treatment times

Fig. 7. Varying the laser dosage and treatment regimes to occlude large blood vessels shows

60x

3

20

Size

of

artery

occlu

ded/(μ

m)

30x

3

30

Treatment time/(s)

1

20

x

4

40

60

80

100

Multiple short laser treatments, interspersed with 'dark' periods, could overcome these limitations by exciting a large number of photosensitizers in a short period, and then allowing the blood flow to replenish the ground state photosensitizer and/or oxygen during the 'dark' period. The examples in Fig. 7 and 8 clearly indicate that multiple short laser treatments are more efficacious than single long treatments to achieve long-term occlusion of large vessels. We were able to completely occlude up to 100μm sized arteries using laser power as low as 70mW with 30sx3 laser treatments, whereas a laser power of 120mW and 120s treatment time only succeeded in occluding up to 60μm arteries. Our data suggest that in the context of clinical verteporfin TPE-PDT treatment, it would be useful to investigate multiple short laser treatments to achieve optimal PDT efficiency and shut down large

that multiple short treatments are more useful than long single treatments in vessel

are preferred.

occlusion.

60

laser

80

power/(mW)

100

120

140

160

1

20x2

feeder vessels.

#### **3.2.1 The mouse window chamber model for testing vascular occlusion**

Khurana et al (Khurana et al., 2009) have investigated TPE-PDT in a murine chamber window model. Surgical placement of a transparent window (1cm diameter) into the dorsal skin of a mouse allows for direct visualization of skin vasculature and administration of PDT treatment under a confocal laser scanning microscope (see Figure 9). This has the advantage of using a more robust animal model that allows for tracking of vascular occlusion on the long term - up to 25hrs. With the chicken CAM model, it is typically possible to track vessel occlusion only up to 6-7hrs after the treatment. The disadvantage of using the mouse model for wet-AMD is that the blood vessels tested are normal healthy vessels, not leaky neovasculature of the type found in wet-AMD and the chicken CAM.

Fig. 9. The murine window chamber model. Nude mouse with surgically implanted window (left), vasculature visible through the window (right) [Reproduced with permission from Khurana et al., (2009) *Journal of Biomedical Optics*, 14, 064006]

Two-Photon Excitation Photodynamic

**5. TPE-PDT and anti-VEGF therapy** 

neovasculature is therefore, strongly indicated.

the retinal macula and destroying pre-existing leakage.

Chiang & Regillo, 2011).

**6. Conclusions** 

Therapy: Working Toward a New Treatment for Wet Age-Related Macular Degeneration 223

The next challenge would be to develop adaptive optics for localized delivery of laser light into the retinal neovasculature. The laser beam needs to be tightly focused upon the macular neovasculature, and the defocusing effect of the eye-lens as well as the cornea needs to be overcome to achieve this. Research is currently under way at the Campbell laboratory,

Photodynamic therapy has proved extremely useful in prohibiting choroidal neovascular leakage and conservation of visual acuity (Arnold et al., 2001; Azab et al., 2004). It does not, however, prevent reoccurrence of retinal leakage, and treatment needs to be repeated at regular intervals (Schmidt-Erfurth et al., 1999). For effective arrest and/or cure of wet-AMD, PDT needs to be complemented by other treatments that prevent the formation of neovasculature. Of these, anti-VEGF therapy shows the greatest promise (Abouammoh & Sharma, 2011; Chiang & Regillo, 2011; Ozkiris, 2010). It targets VEGF-A, the vascular endothelial growth factor (VEGF) that is associated with promoting neovascularisation and angiogenesis. The role of VEGF-A in the pathogenesis of the wet-AMD is well recognized (Ferrara et al., 2003; Kliffen et al., 1997). PDT has also been implicated in the upregulation of VEGF, thereby promoting vascularisation, even as it eradicates the existing neovasculature. A treatment strategy that targets VEGF at the same time as it occludes existing

Currently, two anti-VEGF strategies are most commonly used in treatment, both of which involve antibodies to VEGF-A (Abouammoh & Sharma, 2011). Bevacizumab (Avastin, Genentech, San Fransisco, California, USA) is a humanized monoclonal antibody to VEGF-A, while Ranibizumab (Lucentis, Genentech, San Fransisco, California, USA) is a monoclonal antibody fragment derived from Bevacizumab. Both Avastin and Lucentis are administered through intraocular injections and have been shown to significantly improve the visual acuity. In both cases, the treatment requires repeated intraocular injections and neither of them can eradicate pre-existing neovasculature. Combining anti-VEGF therapy with TPE-PDT for treating wet-AMD has the potential to reduce the number of anti-VEGF injections, while still greatly improving the visual acuity by blocking neovascularisation of

Preliminary investigations of combined Avastin and one-photon PDT treatments suggest that while the combination therapy does not always improve visual acuity, it does reduce the number of retreatments required (Abouammoh & Sharma, 2011; Chiang & Regillo, 2011). Clinical trials are currently under way in North America and Europe to investigate the effects of combined PDT and Lucentis anti-VEGF therapy (Abouammoh & Sharma, 2011;

Laboratory experiments show that two-photon excitation photodynamic therapy offers potential for better selectivity and lower collateral damage in treating wet age-related macular degeneration. Experiments in the chicken embryo and mouse window models show that it is possible to achieve long-term, complete occlusion of blood vessels using twophoton excitation of verteporfin. Design of photosensitizers with higher two-photon

University of Waterloo, to develop an ophthalmoscope for TPE-PDT.

#### **3.2.2 Vessel occlusion by TPE-PDT in the windowed mouse**

Khurana et al (Khurana et al., 2009) tested two different photosensitizers for TPE-PDT – verteporfin and a novel porphyrin dimer specifically designed for larger two-photon absorption. For TPE-PDT, a small area (80x80μm2) on the selected blood vessel was raster scanned with the appropriate laser (865nm for verteporfin and 920nm for the porphyrin dimer). A range of photosensitizer and light doses were investigated in order to obtain the optimal value of drug-light product (product of drug concentration and light fluence) and compare TPE-PDT to conventional one photon excitation PDT. The authors focused on complete occlusion of 40-50µm diameter arteries and compared the verteporfin drug-light product of one and two-photon PDT.

The drug-light product for verteporfin TPE-PDT was more than three orders of magnitude higher than the corresponding value for one photon PDT. This was as expected since verteporfin has a much lower absorption cross-section for two-photon excitation. Consequently, a much higher light and drug dose would be necessary to achieve the same vascular occlusion. Interestingly, the corresponding verteporfin TPE-PDT drug-light product in the chicken CAM was ten times higher, suggesting that the CAM vasculature is much less responsive to TPE-PDT. It must be noted that the neovasculature in the CAM is very leaky compared to the mouse vasculature tested. It is quite possible that the higher drug-light product in the CAM is a consequence of lower effective drug concentrations in the excitation volume due to the leaky nature of the vessels.

The porphyrin dimer (dimer 1, fig.3) tested by Khurana et al (Khurana et al., 2009) had a 340-fold higher two-photon absorption cross-section compared to verteporfin, but exhibited only a twenty times lower drug-light product. The lower than expected effectiveness of the photosensitizer is probably due to poorer uptake and/or different localization to PDTsensitive sites in the vasculature.

#### **4. TPE-PDT: Challenges**

For the transition of TPE-PDT from the lab to the clinic, there are still several roadblocks to be met. The main challenges in the development of TPE-PDT are the need for: TPE-specific drugs, inexpensive lasers, adaptive optics to correct for optical aberration in the lens/cornea, and integration of technology into a "point and shoot" package for physicians.

A good TPE-specific drug needs to have high two-photon absorption, low human toxicity, high efficiency for singlet oxygen generation and should be easily targeted to the retinal neovasculature. The previously mentioned Porphyrin Dimer 1 (section 2 and fig. 3) meets at least three of these criteria. It has high two-photon absorption, very high singlet oxygen yield and shows promise for targeting to the neovasculature (Collins et al., 2008). It is, as such, a promising candidate for testing as a TPE-PDT drug.

Currently, TPE-PDT can only be achieved by excitation with expensive, high energy, pulsed lasers that can provide the high energy densities required for two-photon absorption. However, photosensitizers with very high two-photon absorption can potentially be activated by inexpensive low energy lasers, making TPE-PDT an economically viable treatment. Porphyrin dimers, with their large two-photon absorption cross-section and high efficiency of singlet oxygen generation, hold out the promise of inexpensive TPE-PDT treatment (Drobizhev et al., 2005; Kobuke & Ogawa, 2008).

The next challenge would be to develop adaptive optics for localized delivery of laser light into the retinal neovasculature. The laser beam needs to be tightly focused upon the macular neovasculature, and the defocusing effect of the eye-lens as well as the cornea needs to be overcome to achieve this. Research is currently under way at the Campbell laboratory, University of Waterloo, to develop an ophthalmoscope for TPE-PDT.

#### **5. TPE-PDT and anti-VEGF therapy**

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

Khurana et al (Khurana et al., 2009) tested two different photosensitizers for TPE-PDT – verteporfin and a novel porphyrin dimer specifically designed for larger two-photon absorption. For TPE-PDT, a small area (80x80μm2) on the selected blood vessel was raster scanned with the appropriate laser (865nm for verteporfin and 920nm for the porphyrin dimer). A range of photosensitizer and light doses were investigated in order to obtain the optimal value of drug-light product (product of drug concentration and light fluence) and compare TPE-PDT to conventional one photon excitation PDT. The authors focused on complete occlusion of 40-50µm diameter arteries and compared the verteporfin drug-light

The drug-light product for verteporfin TPE-PDT was more than three orders of magnitude higher than the corresponding value for one photon PDT. This was as expected since verteporfin has a much lower absorption cross-section for two-photon excitation. Consequently, a much higher light and drug dose would be necessary to achieve the same vascular occlusion. Interestingly, the corresponding verteporfin TPE-PDT drug-light product in the chicken CAM was ten times higher, suggesting that the CAM vasculature is much less responsive to TPE-PDT. It must be noted that the neovasculature in the CAM is very leaky compared to the mouse vasculature tested. It is quite possible that the higher drug-light product in the CAM is a consequence of lower effective drug concentrations in

The porphyrin dimer (dimer 1, fig.3) tested by Khurana et al (Khurana et al., 2009) had a 340-fold higher two-photon absorption cross-section compared to verteporfin, but exhibited only a twenty times lower drug-light product. The lower than expected effectiveness of the photosensitizer is probably due to poorer uptake and/or different localization to PDT-

For the transition of TPE-PDT from the lab to the clinic, there are still several roadblocks to be met. The main challenges in the development of TPE-PDT are the need for: TPE-specific drugs, inexpensive lasers, adaptive optics to correct for optical aberration in the lens/cornea, and integration of technology into a "point and shoot" package for physicians. A good TPE-specific drug needs to have high two-photon absorption, low human toxicity, high efficiency for singlet oxygen generation and should be easily targeted to the retinal neovasculature. The previously mentioned Porphyrin Dimer 1 (section 2 and fig. 3) meets at least three of these criteria. It has high two-photon absorption, very high singlet oxygen yield and shows promise for targeting to the neovasculature (Collins et al., 2008). It is, as

Currently, TPE-PDT can only be achieved by excitation with expensive, high energy, pulsed lasers that can provide the high energy densities required for two-photon absorption. However, photosensitizers with very high two-photon absorption can potentially be activated by inexpensive low energy lasers, making TPE-PDT an economically viable treatment. Porphyrin dimers, with their large two-photon absorption cross-section and high efficiency of singlet oxygen generation, hold out the promise of inexpensive TPE-PDT

**3.2.2 Vessel occlusion by TPE-PDT in the windowed mouse** 

the excitation volume due to the leaky nature of the vessels.

such, a promising candidate for testing as a TPE-PDT drug.

treatment (Drobizhev et al., 2005; Kobuke & Ogawa, 2008).

product of one and two-photon PDT.

sensitive sites in the vasculature.

**4. TPE-PDT: Challenges** 

Photodynamic therapy has proved extremely useful in prohibiting choroidal neovascular leakage and conservation of visual acuity (Arnold et al., 2001; Azab et al., 2004). It does not, however, prevent reoccurrence of retinal leakage, and treatment needs to be repeated at regular intervals (Schmidt-Erfurth et al., 1999). For effective arrest and/or cure of wet-AMD, PDT needs to be complemented by other treatments that prevent the formation of neovasculature. Of these, anti-VEGF therapy shows the greatest promise (Abouammoh & Sharma, 2011; Chiang & Regillo, 2011; Ozkiris, 2010). It targets VEGF-A, the vascular endothelial growth factor (VEGF) that is associated with promoting neovascularisation and angiogenesis. The role of VEGF-A in the pathogenesis of the wet-AMD is well recognized (Ferrara et al., 2003; Kliffen et al., 1997). PDT has also been implicated in the upregulation of VEGF, thereby promoting vascularisation, even as it eradicates the existing neovasculature. A treatment strategy that targets VEGF at the same time as it occludes existing neovasculature is therefore, strongly indicated.

Currently, two anti-VEGF strategies are most commonly used in treatment, both of which involve antibodies to VEGF-A (Abouammoh & Sharma, 2011). Bevacizumab (Avastin, Genentech, San Fransisco, California, USA) is a humanized monoclonal antibody to VEGF-A, while Ranibizumab (Lucentis, Genentech, San Fransisco, California, USA) is a monoclonal antibody fragment derived from Bevacizumab. Both Avastin and Lucentis are administered through intraocular injections and have been shown to significantly improve the visual acuity. In both cases, the treatment requires repeated intraocular injections and neither of them can eradicate pre-existing neovasculature. Combining anti-VEGF therapy with TPE-PDT for treating wet-AMD has the potential to reduce the number of anti-VEGF injections, while still greatly improving the visual acuity by blocking neovascularisation of the retinal macula and destroying pre-existing leakage.

Preliminary investigations of combined Avastin and one-photon PDT treatments suggest that while the combination therapy does not always improve visual acuity, it does reduce the number of retreatments required (Abouammoh & Sharma, 2011; Chiang & Regillo, 2011). Clinical trials are currently under way in North America and Europe to investigate the effects of combined PDT and Lucentis anti-VEGF therapy (Abouammoh & Sharma, 2011; Chiang & Regillo, 2011).

### **6. Conclusions**

Laboratory experiments show that two-photon excitation photodynamic therapy offers potential for better selectivity and lower collateral damage in treating wet age-related macular degeneration. Experiments in the chicken embryo and mouse window models show that it is possible to achieve long-term, complete occlusion of blood vessels using twophoton excitation of verteporfin. Design of photosensitizers with higher two-photon

Two-Photon Excitation Photodynamic

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two-photon excitation fluorescence microscopy and other nonlinear imaging

absorption cross sections is likely to improve the efficacy of the TPE-PDT treatment modality in wet-AMD. Pre-clinical and clinical studies on the safety and efficacy of twophoton excitation photodynamic therapy of wet-AMD are needed before two-photon excitation photodynamic therapy is applied for clinical use.

#### **7. References**


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degeneration. *Current Opinion in Ophthalmology,* 22, 199-204.

nanotoxicity. *Chemical Physics Letters,* 488, 99-111.

Fish, G. E., Hao, Y., Haynes, L., Lim, J. I., Menchini, U., Miller, J. W., Mones, J., Potter, M. J., Reaves, A., Rosenfeld, P. J., Strong, A., Su, X. Y., Slakter, J. S., Schmidt-Erfurth, U. & Sorenson, J. A., (2004) Verteporfin therapy of subfoveal choroidal neovascularization in age-related macular degeneration: Meta-analysis of 2-year safety results in three randomized clinical trials: Treatment of age-related macular degeneration with photodynamic therapy and verteporfin in photodynamic

Impact on ophthalmology and visual sciences. *Investigative Ophthalmology and* 

nanoparticles in embryonic blood vessels: Towards a physicochemical basis for

neovascular age-related macular degeneration. *Current Opinion in Ophthalmology,*

Farrow, A., McKay, S., McKechnie, R., Evans, G., Aaberg, T. M., Brower, J., Waldron, R., Loupe, D., Gillman, J., Myles, B., Saperstein, D. A., Schachat, A. P., Bressler, N. M., Bressler, S. B., Nesbitt, P., Porter, T., Hawse, P., Harnett, M., Eager, A., Belt, J., Cain, D., Emmert, D., George, T., Herring, M., McDonald, J., Mones, J., Corcostegui, B., Gilbert, M., Duran, N., Sisquella, M., Nolla, A., Margalef, A., Miller, J. W., Gragoudas, E. S., Lane, A. M., Emmanuel, N., Holbrook, A., Evans, C., Lord, U. S., Walsh, D. K., Callahan, C. D., DuBois, J. L., Moy, J., Kenney, A. G., Milde, I., Platz, E. S., Lewis, H., Kaiser, P. K., Holody, L. J., Lesak, E., Lichterman, S., Siegel, H., Fattori, A., Ambrose, G., Fecko, T., Ross, D., Burke, S., Conway, J., Singerman, L., Zegarra, H., Novak, M., Bartel, M., Tilocco-DuBois, K., Ilc, M., Schura, S., Joyce, S., Tanner, V., Rowe, P., Smith-Brewer, S., Greanoff, G., Daley, G., DuBois, J., Lehnhardt, D., Kukula, D., Fish, G. E., Jost, B. F., Anand, R., Callanan, D., Arceneaux, S., Arnwine, J., Ellenich, P., King, J., Aguado, H., Rollins, R., Anderson, T., Nork, C., Duignan, K., Boleman, B., Jurklies, B., Pauleikhoff, D., Hintzmann, A., Fischer, M., Sowa, C., et al., (2001) Verteporfin therapy of subfoveal choroidal neovascularization in age-related macular degeneration: Twoyear results of a randomized clinical trial including lesions with occult with no classic choroidal neovascularization-verteporfin in photodynamic therapy report 2.

excitation photodynamic therapy is applied for clinical use.

*American Journal of Ophthalmology,* 131, 541-560.

therapy study report no. 4. *Retina,* 24, 1-12.

*Visual Science,* 41, 624-628.

**7. References** 

22, 152-158.


Rattner, A. & Nathans, J., (2006) Macular degeneration: Recent advances and therapeutic opportunities. *Nature Reviews Neuroscience,* 7, 860-872.

**12** 

*Japan* 

**Clinical Application of Drug** 

Noriyuki Kuno and Shinobu Fujii

*Santen Pharmaceutical Co., Ltd.* 

**Delivery Systems for Treating AMD** 

Due to transparent ocular media, it is relatively easy to observe intraocular tissues such as the vitreous and retina without invasion, and various administration approaches including intravitreal and subretinal injection, or implantation can be applicable. Since the eye-ball is a closed organ, novel therapeutic molecules such as an antisense oligonucleotide for cytomegalovirus retinitis (Fomivirsen; Vitraven®, Isis Pharmaceuticals, Inc., Carlsbad, CA U.S. and Novartis, Basel, Switzerland), an aptamer (e.g. Pegaptanib sodium; Macugen®, Pfizer, Inc., New York, NY, U.S.) or a small interfering RNA for neovascular (wet) agerelated macular degeneration (AMD), have been investigated in human eyes before their applications for systemic diseases. In addition, many injectable or implantable drug delivery systems for chronic vitreoretinal diseases including AMD, diabetic macular edema, retinal vein occlusion, uveitis, and retinitis pigmentosa (RP), using polymer technology and/or

This chapter focuses on drug delivery systems under clinical applications and in late

For the treatment of wet AMD, a standard therapy is monthly intravitreal injections of ranibizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody fragment (Lucentis®, Genentech, Inc., South San Francisco, CA, U.S.) (Genentech Inc.) and photodynamic therapy (PDT) by systemic administration of verteporfin (Visudyne®, QLT Ophthalmics, Inc., Menlo Park, CA, U.S.). The monthly cost of Lucentis® is about \$2,000 and that means effective treatment by Lucentis® faces a serious social problem (Martin et al., 2010, Gower et al., 2010, Patel et al., 2010). Also frequent intravitreal injections might cause several complications, it has been reported that prevalence of lens damage, endophthalmitis and rhegmatogenous retinal detachment were 0.006% (2 of 32,318 injections) (Meyer, Rodrigues et al., 2010), 0.029% (3 of 10,254 cases) (Pilli et al., 2008) and 0.013% (5 of 35,942 injections) (Meyer, Michels et al., 2010), respectively. In addition, recently sustained elevation of intraocular pressure (IOP) after intravitreal injections of anti-VEGF agents has been reported (Good et al., 2011). Although the mechanism of IOP elevation is unclear, aggregation of proteins and/or leaching of silicone from the syringe barrel and rubber stopper might cause to clog the trabecular meshwork. It has also been demonstrated that

experimental stage for the treatment of both wet and atrophic (dry) AMD (Table 1).

mechanical engineering, have been developing (Figure 1).

**2. Significance of drug delivery systems for AMD** 

**1. Introduction** 


## **Clinical Application of Drug Delivery Systems for Treating AMD**

Noriyuki Kuno and Shinobu Fujii *Santen Pharmaceutical Co., Ltd. Japan* 

#### **1. Introduction**

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

Rattner, A. & Nathans, J., (2006) Macular degeneration: Recent advances and therapeutic

Reinke, M. H., Canakis, C., Husain, D., Michaud, N., Flotte, T. J., Gragoudas, E. S. & Miller,

Samkoe, K. S., Fecica, M. S., Goyan, R. L., Buchholz, J. L., Campbell, C., Kelly, N. M. &

Samkoe, K. S., Clancy, A. A., Karotki, A., Wilson, B. C. & Cramb, D. T., (2007) Complete

Samkoe, K. S. (2007) Two-photon excitation photodynamic therapy: Progress towards a new

Schlatter, P., Konig, M. F., Karlsson, L. M. & Burri, P. H., (1997) Quantitative study of

Schmidt-Erfurth, U., Miller, J. W., Sickenberg, M., Laqua, H., Barbazetto, I., Gragoudas, E. S.,

Schmidt-Erfurth, U. & Hasan, T., (2000) Mechanisms of action of photodynamic therapy

Skovsen, E., Snyder, J. W., Lambert, J. D. & Ogilby, P. R., (2005) Lifetime and diffusion of singlet oxygen in a cell. *Journal of Physical Chemistry B,* 109, 8570-8573. So, P. T. C., Dong, C. Y., Masters, B. R. & Berland, K. M., (2000) Two-photon excitation fluorescence microscopy. *Annual Review of Biomedical Engineering,* 2, 399-429. Soubrane, G. & Bressler, N. M., (2001) Treatment of subfoveal choroidal neovascularisation

photodynamic therapy. *British Journal of Ophthalmology,* 85, 483-495.

choroid in the cynomolgus monkey. *Ophthalmology,* 106, 1915-1923.

macular degeneration. *Journal of Biomedical Optics,* 12, 034025.

chicken embryo. *Microvascular Research,* 54, 65-73.

and 2 study. *Archives of Ophthalmology,* 117, 1177-1187.

Department of Chemistry, University of Calgary, Calgary, Canada.

J. W., (1999) Verteporfin photodynamic therapy retreatment of normal retina and

Cramb, D. T., (2006) Photobleaching kinetics of optically trapped multilamellar vesicles containing verteporfin using two-photon excitation. *Photochemistry and* 

blood vessel occlusion in the chick chorioallantoic membrane using two-photon excitation photodynamic therapy: Implications for treatment of wet age-related

treatment for wet age-related macular degeneration. PhD thesis submitted to the

intussusceptive capillary growth in the chorioallantoic membrane (cam) of the

Zografos, L., Piguet, B., Pournaras, C. J., Donati, G., Lane, A. M., Birngruber, R., van den Berg, H., Strong, H. A., Manjuris, U., Gray, T., Fsadni, M. & Bressler, N. M., (1999) Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration: Results of retreatments in a phase 1

with verteporfin for the treatment of age-related macular degeneration. *Survey of* 

in age related macular degeneration: Focus on clinical application of verteporfin

opportunities. *Nature Reviews Neuroscience,* 7, 860-872.

*Photobiology,* 82, 152-157.

*Ophthalmology,* 45, 195-214.

Due to transparent ocular media, it is relatively easy to observe intraocular tissues such as the vitreous and retina without invasion, and various administration approaches including intravitreal and subretinal injection, or implantation can be applicable. Since the eye-ball is a closed organ, novel therapeutic molecules such as an antisense oligonucleotide for cytomegalovirus retinitis (Fomivirsen; Vitraven®, Isis Pharmaceuticals, Inc., Carlsbad, CA U.S. and Novartis, Basel, Switzerland), an aptamer (e.g. Pegaptanib sodium; Macugen®, Pfizer, Inc., New York, NY, U.S.) or a small interfering RNA for neovascular (wet) agerelated macular degeneration (AMD), have been investigated in human eyes before their applications for systemic diseases. In addition, many injectable or implantable drug delivery systems for chronic vitreoretinal diseases including AMD, diabetic macular edema, retinal vein occlusion, uveitis, and retinitis pigmentosa (RP), using polymer technology and/or mechanical engineering, have been developing (Figure 1).

This chapter focuses on drug delivery systems under clinical applications and in late experimental stage for the treatment of both wet and atrophic (dry) AMD (Table 1).

#### **2. Significance of drug delivery systems for AMD**

For the treatment of wet AMD, a standard therapy is monthly intravitreal injections of ranibizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody fragment (Lucentis®, Genentech, Inc., South San Francisco, CA, U.S.) (Genentech Inc.) and photodynamic therapy (PDT) by systemic administration of verteporfin (Visudyne®, QLT Ophthalmics, Inc., Menlo Park, CA, U.S.). The monthly cost of Lucentis® is about \$2,000 and that means effective treatment by Lucentis® faces a serious social problem (Martin et al., 2010, Gower et al., 2010, Patel et al., 2010). Also frequent intravitreal injections might cause several complications, it has been reported that prevalence of lens damage, endophthalmitis and rhegmatogenous retinal detachment were 0.006% (2 of 32,318 injections) (Meyer, Rodrigues et al., 2010), 0.029% (3 of 10,254 cases) (Pilli et al., 2008) and 0.013% (5 of 35,942 injections) (Meyer, Michels et al., 2010), respectively. In addition, recently sustained elevation of intraocular pressure (IOP) after intravitreal injections of anti-VEGF agents has been reported (Good et al., 2011). Although the mechanism of IOP elevation is unclear, aggregation of proteins and/or leaching of silicone from the syringe barrel and rubber stopper might cause to clog the trabecular meshwork. It has also been demonstrated that

Clinical Application of Drug Delivery Systems for Treating AMD 229

**PEGylation** 

**Sustained release** 

**Mode of action** 

aptamer

Anti-C5 aptamer

aptamer

Inhibition of microglial activation

(CNTF)

agonist

NK cell-mediated apoptosis

apoptosis

by PEDF

Antiangiogenesis by endostatin, angiostatin

**Gene induction** 

Ad, adenovirus; AAV2, adeno-associated virus serotype 2; AMD, age-related macular degeneration; C5, complement factor 5; CNTF, ciliary neurotrophic factor; CTLs, cytotoxic T lymphocytes; EIAV, equine infectious anaemia virus; IV, intravenous; IVT, intravitreal; NK, natural killer;

factor; PEG, poly(ethylene gycol); PLGA, poly(lactide-co-glycolide); PVA, poly(vinyl alcohol);

SC, subcutaneous; SRT, subretinal; VEGF, vascular endothelial growth factor Table 1. Promising drug candidates for wet/dry AMD in clinical trials

PDGF, platelet-derived growth factor; PDT, photodynamic therapy; PEDF, pigment epithelium-derived

**Targeting** 

**Administration Route** 

IVT

IVT

IVT

IVT

IVT implant

IVT

injection

IVT

SC

IVT

SRT

injection PEG

injection PEG

injection PEG

implant Polyimide/PVA

implant PLGA

injection -

injection -

injection -

injection Ad

injection AAV2

injection EIAV

Semi-permeable membrane

> Negativelycharged liposome

**Excipients/ Carriers** 

**Active ingredient** 

Fluocinolone

VEGFR epitope peptide

Endstatin

**Brand name** 

ARC1905 - P1 (dAMD)

acetonide Iluvien® P2 (dAMD)

I-con1 - P1/2a

Angiostatin RetinoStat® P1 (wAMD)

**Development stage** 

P1 (wAMD)

P2 (wAMD)

Verteporfin Visudyne® Launched PDT IV

WST-11 Stakel® P2 (wAMD) PDT IV


sFLT01 - P1 (wAMD) VEGF decoy IVT

(wAMD)

PEDF - P1 (wAMD) Antiangiogenesis

NT-501 - P2 (dAMD) Neurotrophin

Brimonidine - P2 (dAMD) <sup>α</sup>2 adrenergic

Pegaptanib Macugen® Launched Anti-VEGF

E10030 - P2 (wAMD) Anti-PDGF

aggregated proteins induce a more significant immunological response than non-aggregated proteins (Rosenberg, 2006). Furthermore, a lack of selective targeting of verteporfin to neovascular endothelial cells causes to damage the normal retinal tissues such as the retinal pigment epithelium (RPE) and photoreceptors. Therefore, it is necessary to develop drug delivery systems which can be easily and non-invasively administered, have long-term controlled-release by a single administration, and/or selective-targeting potency to the pathologic tissues for the treatment of AMD to overcome the disadvantages in the current wet AMD therapy.

Fig. 1. Example of drug delivery systems for the treatment of AMD

On the other hand, tachyphylaxis is a diminished therapeutic response to a drug after repeated administrations over time. It has been reported that 8.5% (5 of 59 patients) of wet AMD patients who received repeated intravitreal injections of bevacizumab developed tachyphylaxis (Forooghian et al., 2009). The median time to develop tachyphylaxis after the first bevacizumab injection was 100 weeks with a median of 8 injections before tachyphylaxis development. Other groups have also reported that tachyphylaxis with ranibizumab and bevacizumab for wet AMD (Schaal et al., 2008, Keane et al., 2008, Eghoj & Sorensen, 2011) was found. It is thought that the generation of neutralizing antibodies to bevacizumab did not significantly contribute to the development of tachyphylaxis (Forooghian et al., 2011). A combination of bevacizumab and triamcinolone acetonide (TA) improved the reduction of bevacizumab efficacy caused by anti-VEGF tachyphylaxis. Therefore, there is an urgent need to develop drugs and their drug delivery systems targeting other pathways not involving VEGF for patients who develop anti-VEGF tachyphylaxis or non-responders.

In addition to wet AMD, dry AMD is a chronic, progressive retinal degenerative disease, therefore, drug delivery systems are absolutely needed.

aggregated proteins induce a more significant immunological response than non-aggregated proteins (Rosenberg, 2006). Furthermore, a lack of selective targeting of verteporfin to neovascular endothelial cells causes to damage the normal retinal tissues such as the retinal pigment epithelium (RPE) and photoreceptors. Therefore, it is necessary to develop drug delivery systems which can be easily and non-invasively administered, have long-term controlled-release by a single administration, and/or selective-targeting potency to the pathologic tissues for the treatment of AMD to overcome the disadvantages in the current

Fig. 1. Example of drug delivery systems for the treatment of AMD

tachyphylaxis or non-responders.

therefore, drug delivery systems are absolutely needed.

On the other hand, tachyphylaxis is a diminished therapeutic response to a drug after repeated administrations over time. It has been reported that 8.5% (5 of 59 patients) of wet AMD patients who received repeated intravitreal injections of bevacizumab developed tachyphylaxis (Forooghian et al., 2009). The median time to develop tachyphylaxis after the first bevacizumab injection was 100 weeks with a median of 8 injections before tachyphylaxis development. Other groups have also reported that tachyphylaxis with ranibizumab and bevacizumab for wet AMD (Schaal et al., 2008, Keane et al., 2008, Eghoj & Sorensen, 2011) was found. It is thought that the generation of neutralizing antibodies to bevacizumab did not significantly contribute to the development of tachyphylaxis (Forooghian et al., 2011). A combination of bevacizumab and triamcinolone acetonide (TA) improved the reduction of bevacizumab efficacy caused by anti-VEGF tachyphylaxis. Therefore, there is an urgent need to develop drugs and their drug delivery systems targeting other pathways not involving VEGF for patients who develop anti-VEGF

In addition to wet AMD, dry AMD is a chronic, progressive retinal degenerative disease,

wet AMD therapy.


Ad, adenovirus; AAV2, adeno-associated virus serotype 2; AMD, age-related macular degeneration; C5, complement factor 5; CNTF, ciliary neurotrophic factor; CTLs, cytotoxic T lymphocytes; EIAV, equine infectious anaemia virus; IV, intravenous; IVT, intravitreal; NK, natural killer; PDGF, platelet-derived growth factor; PDT, photodynamic therapy; PEDF, pigment epithelium-derived factor; PEG, poly(ethylene gycol); PLGA, poly(lactide-co-glycolide); PVA, poly(vinyl alcohol); SC, subcutaneous; SRT, subretinal; VEGF, vascular endothelial growth factor

Table 1. Promising drug candidates for wet/dry AMD in clinical trials

Clinical Application of Drug Delivery Systems for Treating AMD 231

was reported that the 5-HT1A agonists delayed the progression of motor neuron degeneration in pmn mice (Duong et al., 1998) and reduced lipid peroxidation in a rat epilepsy model (de Freitas et al., 2010). Such neuroprotective effects are considered to be caused by neuronal membrane hyperpolarization via G protein-coupled K+ channels, decreasing glutamate release, blocking Ca2+ channels or Na+ channels, activation of MAPK (mitogen-actiated protein kinase)/ERK (extracellular signal-regulated kinase) signalling pathway resulting expression of anti-apoptotic proteins and inhibition of caspase, and an expression of brain derived neurotrophic factor (BDNF) mRNA, S100β and nerve growth

factor. Also 5-HT1A was expressed in rats and rabbits retina (Kusol & Brunken, 2000).

also disclosed.

Fig. 2. Complement cascade

Recently, it has been reported that tandospirone, 5-HT1A agonist, which is widely used for the treatment of anxiety disorders, has a neuroprotective effect for retinal lesions due to light-damage (Collier et al., 2009, Rhoades et al., 2009, Wang et al., 2009, Collier et al., 2010). The studies have also suggested tandospirone increases of MEK (mitogen-activated extracellular signal regulated kinase) 1/2 and ERK 1/2 phosphorylation, leading to the subsequent upregulation of anti-oxidant and anti-apoptotic proteins, including superoxide dismutase (SOD)-1, SOD-2, B-cell lymphoma (Bcl)-2 and Bcl-XL (Rhoades et al., 2009), or a decrease complement factors (C3, CFB, CFH) and membrane attack complex (MAC) deposition in the outer retina (Wang et al., 2009) (Figure 2). Currently, an eye-drops formulation of tandospirone (AL-8309B, Alcon Laboratories, Inc., Fort Worth, TX, U.S.) is under a Phase III study for the treatment of dry AMD (ClinicalTrials.gov. NCT00890097). Actual eye-drop formulation of tandospirone currently conducted in clinical study is not

#### **3. Traditional formulation**

An eye-drop, irrespective of the instilled volume, often eliminates rapidly within 5 to 6 minutes after an administration, and only a small amount (1–3%) of an eye-drop actually reaches the intraocular tissue. Therefore, it is difficult to provide and maintain an adequate concentration of drug in the precorneal area. More than 75% of applied ophthalmic solution is lost via nasolachrymal drainage and absorbed systemically via conjunctiva, then ocular drug availability is very low (Kuno & Fujii, 2011b). Generally topical applied drugs do not reach the posterior segment of the eye, thus, some additives or carriers to enhance the retention time and intraocular absorption are needed. Several eye-drops formulations are challenged to treat for AMD under clinical trials.

#### **3.1 Pazopanib**

Some studies have suggested that inhibition of VEGF signalling alone is sufficient to suppress choroidal neovascularization (CNV), however, others have demonstrated a more potent suppression of angiogenesis by inhibiting multiple tyrosine kinase receptors (Bergers et al., 2003, Erber et al., 2004, Kwak et al., 2000). It may be a more desirable therapeutic approach that drugs inhibit multiple angiogenic pathways. Pazopanib is a multi-tyrosine kinase inhibitor of VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor (PDGFR)-α and -β, fibroblast growth factor receptor (FGFR) -1 and -3, cytokine receptor (Kit), interleukin-2 receptor inducible T-cell kinase (Itk), leukocyte-specific protein tyrosine kinase (Lck), and transmembrane glycoprotein receptor tyrosine kinase (c-Fms). *In vitro*, pazopanib inhibited ligand-induced autophosphorylation of VEGFR-2, Kit and PDGFR-β receptors. *In vivo*, pazopanib inhibited VEGF-induced VEGFR-2 phosphorylation in mouse lungs, angiogenesis in a mouse model, and the growth of some human tumor xenografts in mice (GlaxoSmithKline plc.). Pazopanib is currently prescribed for advanced renal cell carcinoma.

Yafai et al. have demonstrated that eye-drop formulation of pazopanib complexed with cyclodextrin significantly inhibited CNV in laser-induced CNV rat model (Yafai et al., 2011). Since this effect was obtained by overdose of eye-drops (30 μL/eye), it is doubtful whether this effective inhibition of CNV resulted from a topical absorption of pazopanib. A phase II clinical study of pazopanib eye-drops for the treatment of wet AMD is currently underway (ClinicalTrials.gov. NCT01134055). Unfortunately, actual formulation of pazopanib eyedrops used in clinical study is not disclosed.

#### **3.2 Tandospirone**

Serotonin (5-hydroxytryptamine; 5-HT) and its multiple receptors regulate various physiological functions. 5-HT1A receptor plays an important role for the control of sleep, feeding and anxiety. 5-HT1A receptor agonists have also neuroprotective effects in animal models including central nervous system ischemia (Saruhashi et al., 2002, Mauler & Horvath, 2005, Ramos et al., 2004, Kukley et al., 2001, Torup et al., 2000, Piera et al., 1995), acute subdermal hematoma (Fournier et al., 1993), traumatic brain injury (Alessandri et al., 1999, Kline et al., 2002), excitotoxicity (Oosterink et al., 2003, Cosi et al., 2005), a Parkinson's disease model animal induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Bibbiani et al., 2001, Bezard et al., 2006), and sciatic nerve crush (Fournier et al., 1993). Additionally, it

An eye-drop, irrespective of the instilled volume, often eliminates rapidly within 5 to 6 minutes after an administration, and only a small amount (1–3%) of an eye-drop actually reaches the intraocular tissue. Therefore, it is difficult to provide and maintain an adequate concentration of drug in the precorneal area. More than 75% of applied ophthalmic solution is lost via nasolachrymal drainage and absorbed systemically via conjunctiva, then ocular drug availability is very low (Kuno & Fujii, 2011b). Generally topical applied drugs do not reach the posterior segment of the eye, thus, some additives or carriers to enhance the retention time and intraocular absorption are needed. Several eye-drops formulations are

Some studies have suggested that inhibition of VEGF signalling alone is sufficient to suppress choroidal neovascularization (CNV), however, others have demonstrated a more potent suppression of angiogenesis by inhibiting multiple tyrosine kinase receptors (Bergers et al., 2003, Erber et al., 2004, Kwak et al., 2000). It may be a more desirable therapeutic approach that drugs inhibit multiple angiogenic pathways. Pazopanib is a multi-tyrosine kinase inhibitor of VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor (PDGFR)-α and -β, fibroblast growth factor receptor (FGFR) -1 and -3, cytokine receptor (Kit), interleukin-2 receptor inducible T-cell kinase (Itk), leukocyte-specific protein tyrosine kinase (Lck), and transmembrane glycoprotein receptor tyrosine kinase (c-Fms). *In vitro*, pazopanib inhibited ligand-induced autophosphorylation of VEGFR-2, Kit and PDGFR-β receptors. *In vivo*, pazopanib inhibited VEGF-induced VEGFR-2 phosphorylation in mouse lungs, angiogenesis in a mouse model, and the growth of some human tumor xenografts in mice (GlaxoSmithKline plc.). Pazopanib is currently prescribed

Yafai et al. have demonstrated that eye-drop formulation of pazopanib complexed with cyclodextrin significantly inhibited CNV in laser-induced CNV rat model (Yafai et al., 2011). Since this effect was obtained by overdose of eye-drops (30 μL/eye), it is doubtful whether this effective inhibition of CNV resulted from a topical absorption of pazopanib. A phase II clinical study of pazopanib eye-drops for the treatment of wet AMD is currently underway (ClinicalTrials.gov. NCT01134055). Unfortunately, actual formulation of pazopanib eye-

Serotonin (5-hydroxytryptamine; 5-HT) and its multiple receptors regulate various physiological functions. 5-HT1A receptor plays an important role for the control of sleep, feeding and anxiety. 5-HT1A receptor agonists have also neuroprotective effects in animal models including central nervous system ischemia (Saruhashi et al., 2002, Mauler & Horvath, 2005, Ramos et al., 2004, Kukley et al., 2001, Torup et al., 2000, Piera et al., 1995), acute subdermal hematoma (Fournier et al., 1993), traumatic brain injury (Alessandri et al., 1999, Kline et al., 2002), excitotoxicity (Oosterink et al., 2003, Cosi et al., 2005), a Parkinson's disease model animal induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Bibbiani et al., 2001, Bezard et al., 2006), and sciatic nerve crush (Fournier et al., 1993). Additionally, it

**3. Traditional formulation** 

**3.1 Pazopanib** 

challenged to treat for AMD under clinical trials.

for advanced renal cell carcinoma.

**3.2 Tandospirone** 

drops used in clinical study is not disclosed.

was reported that the 5-HT1A agonists delayed the progression of motor neuron degeneration in pmn mice (Duong et al., 1998) and reduced lipid peroxidation in a rat epilepsy model (de Freitas et al., 2010). Such neuroprotective effects are considered to be caused by neuronal membrane hyperpolarization via G protein-coupled K+ channels, decreasing glutamate release, blocking Ca2+ channels or Na+ channels, activation of MAPK (mitogen-actiated protein kinase)/ERK (extracellular signal-regulated kinase) signalling pathway resulting expression of anti-apoptotic proteins and inhibition of caspase, and an expression of brain derived neurotrophic factor (BDNF) mRNA, S100β and nerve growth factor. Also 5-HT1A was expressed in rats and rabbits retina (Kusol & Brunken, 2000).

Recently, it has been reported that tandospirone, 5-HT1A agonist, which is widely used for the treatment of anxiety disorders, has a neuroprotective effect for retinal lesions due to light-damage (Collier et al., 2009, Rhoades et al., 2009, Wang et al., 2009, Collier et al., 2010). The studies have also suggested tandospirone increases of MEK (mitogen-activated extracellular signal regulated kinase) 1/2 and ERK 1/2 phosphorylation, leading to the subsequent upregulation of anti-oxidant and anti-apoptotic proteins, including superoxide dismutase (SOD)-1, SOD-2, B-cell lymphoma (Bcl)-2 and Bcl-XL (Rhoades et al., 2009), or a decrease complement factors (C3, CFB, CFH) and membrane attack complex (MAC) deposition in the outer retina (Wang et al., 2009) (Figure 2). Currently, an eye-drops formulation of tandospirone (AL-8309B, Alcon Laboratories, Inc., Fort Worth, TX, U.S.) is under a Phase III study for the treatment of dry AMD (ClinicalTrials.gov. NCT00890097). Actual eye-drop formulation of tandospirone currently conducted in clinical study is not also disclosed.

Fig. 2. Complement cascade

Clinical Application of Drug Delivery Systems for Treating AMD 233

times compared to the parent drug (non-PEGylated aptamer) (Simone Fishburn, 2008). After an intravitreal injection, pegaptanib is absorbed intact into the systemic circulation, but the concentration in plasma was 800-several thousand-fold lower than that in the vitreous. In addition, the elimination half-life was 9.3 hours after a single intravenous injection in rhesus monkeys (1 mg/kg). This "flip-flop" kinetics might cause to estimate the vitreous humor half-life in the vitreous from the plasma half-life in human. In clinical situation, pegaptanib

ARC1905 (Ophthotech Corp., Princeton, NJ, U.S.) is a chemically-modified oligonucleotide of 39 nucleotides bound to branched PEG (two arms of 20 kDa linear PEG units), and binds to complement factor C5, leading to prevent the formation of key terminal fragments C5a and MAC (C5b-9). C5a is an important inflammatory activator inducing vascular permeability, recruitment and activation of phagocytes. MAC is involved to initiate cell lysis. Therefore, by inhibiting these C5-mediated inflammation and RPE death leading to geographic atrophy (GA), ARC1905 might be promising for both wet and dry AMD (Kuno & Fujii, 2011a). A phase I study to evaluate the safety, tolerability, and pharmacokinetic profile of multiple doses of intravitreal ARC1905 in combination with multiple doses of Lucentis® is currently in progress. In addition, it has been demonstrated by histopathological examination human dry AMD lesions strongly stained for C5a and MAC at key pathology sites (Anderson et al., 2002). A Phase I clinical trial to evaluate of an intravitreal ARC1905 in patients with GA is undergoing (ClinicalTrials.gov. NCT00950638).

E10030 (Ophthotech Corp.) is a chemically-modified oligonucleotide of 29 nucleotides linked with branched PEG (two arms of 20 kDa linear PEG units), and binds to PDGF-B, which is known to play a role of in the recruitment and maturation of pericytes that can increase resistance to the anti-VEGF treatment for wet AMD. PDGF and its receptor (PDGFR) do not act on vascular endothelial cells, but on pericytes. Therefore, inhibition of PDGF signalling might cause to achieve regression of neovascular vessels. Jo et al. have demonstrated that a combination therapy with anti-VEGF aptamer (Pegaptanib sodium) and anti-PDGFR-β antibody is more effective for CNV prevention and regression compared to monotherapy in the laser-induced CNV model (Jo et al., 2006). In an open-label Phase I clinical study conducted by Ophthotech, 59% of patients treated with E10030 and Lucentis® gained significant vision (3-line gain or better) at 12 weeks after the start of therapy. Interestingly, there was a mean decrease of 86% in the area of CNV at 12 weeks (Ophthotech Corporation). A randomized, controlled, Phase II study of E10030 in combination with Lucentis® for the treatment of wet AMD is currently underway (ClinicalTrials.gov.

To reduce the frequency of administration, many controlled drug delivery systems have been investigated by using biodegradable or non-biodegradable polymeric devices for the treatment of various retinal diseases as well as AMD (Kuno & Fujii, 2010). In general, drug release from biodegradable matrices consisting of poly(lactide-co-glycolide) (PLGA) is

is used as intravitreal injections of 0.3 mg once every 6 weeks.

**4.2 ARC1905** 

**4.3 E10030** 

NCT01089517).

**5. Sustained-release systems** 

In the classical pathway, the cascade is initiated by the binding of C1q to antibody-antigen complex. The lectin pathway is initiated by the binding of carbohydrates associated with microbes to lectin proteins such as mannose-binding lectin (MBL). C1q and MBL form complexes with mannose-binding lectin-associated serine protease (MASP), which cleave C4 into C4a and C4b,C2 into C2a and C2b. C4b binds to C2a (C4bC2a), work as a C3 convertase resulting degradation of C3 into C3a and C3b. C3b binds with C4bC2a to form C4bC2aC3b work as a C5 convertases.

In the alternative pathway, C3 convertase is formed via a spontaneous hydrolysis of an internal C3 thioester into C3(H2O). C3(H2O) binds to factor B and D and forms soluble C3 convertase; C3(H2O)Bb and subsequently formed membrane-bound C3 convertase; C3bBb resulting cleavage of C3 into C3a and C3b. C3b binds C3bBb to form C3bBbC3b (C5 convertase).

In all pathways, C5 convertases cleaves C5 to C5a and C5b. C5b initiates the formation of the membrane attack complex (MAC) consisting of C5b, C6, C7, C8, and C9. The MAC creates a pore in the cell membrane of its targets (microbes, damaged cells) leading to cell lysis and death. The anaphylatoxins C3a and C5a work to increase vascular permeability, initiate degranulation of mast cells and neutrophils, induce cytokine release from macrophages, and mediate leukocyte chemotaxis.

Complement factor H (CFH) inhibits C3b through complement factor I (CFI) binding. Clusterin and vitronection (Vn) inhibits MAC formation by binding with a complex of C5b-7.

### **4. PEGylation**

Covalent bonding of drug molecules to poly(ethylene glycol) (PEG), referred to as PEGylation, is a popular approach to modify and enhance the water solubility and pharmacokinetic and pharmacodynamic properties of biological and small-molecule drugs. In general, PEGs are inert water-soluble polymers, but recently it has been reported that a subretinal injection of PEG induced CNV with dose-dependency via complement activation in mice (Lyzogubov et al., 2011). PEGs can be attached to proteins and other therapeutic molecules, leading to increase the hydrodynamic volume of the therapeutic molecules. In addition, PEGs can shield drugs from interactions with enzymes and from inactivation by the immune system. As a result, PEGylated drugs can exhibit prolonged half-life, higher stability, increased water solubility, and reduced immunogenicity. It is thought that conjugates bearing branched chain PEG show increased thermal stability and higher resistance to enzymatic degradation compared to bearing linear PEG (Hamidi et al., 2008).

#### **4.1 Macugen®**

Pegaptanib sodium is a chemically-modified oligonucleotide of 28 nucleotides which linked with 40 kDa branched PEG (two arms of 20 kDa linear PEG units), which binds to VEGF165. Pegaptanib was approved by FDA in 2004, and was both the first approved aptamer-based drug and the first approved pharmacotherapy for wet AMD. In rabbit eyes at 24 hours after an intravitreal injection, radiolabeled pegaptanib could be penetrated and distributed in the retina (Eyetech Inc.). In a monkey pharmacokinetics study, pegaptanib was eliminated from the vitreous with a half-life of 94 hours (Drolet et al., 2000), which has been increased by 3.91 times compared to the parent drug (non-PEGylated aptamer) (Simone Fishburn, 2008). After an intravitreal injection, pegaptanib is absorbed intact into the systemic circulation, but the concentration in plasma was 800-several thousand-fold lower than that in the vitreous. In addition, the elimination half-life was 9.3 hours after a single intravenous injection in rhesus monkeys (1 mg/kg). This "flip-flop" kinetics might cause to estimate the vitreous humor half-life in the vitreous from the plasma half-life in human. In clinical situation, pegaptanib is used as intravitreal injections of 0.3 mg once every 6 weeks.

#### **4.2 ARC1905**

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

In the classical pathway, the cascade is initiated by the binding of C1q to antibody-antigen complex. The lectin pathway is initiated by the binding of carbohydrates associated with microbes to lectin proteins such as mannose-binding lectin (MBL). C1q and MBL form complexes with mannose-binding lectin-associated serine protease (MASP), which cleave C4 into C4a and C4b,C2 into C2a and C2b. C4b binds to C2a (C4bC2a), work as a C3 convertase resulting degradation of C3 into C3a and C3b. C3b binds with C4bC2a to form C4bC2aC3b

In the alternative pathway, C3 convertase is formed via a spontaneous hydrolysis of an internal C3 thioester into C3(H2O). C3(H2O) binds to factor B and D and forms soluble C3 convertase; C3(H2O)Bb and subsequently formed membrane-bound C3 convertase; C3bBb resulting cleavage of C3 into C3a and C3b. C3b binds C3bBb to form C3bBbC3b (C5

In all pathways, C5 convertases cleaves C5 to C5a and C5b. C5b initiates the formation of the membrane attack complex (MAC) consisting of C5b, C6, C7, C8, and C9. The MAC creates a pore in the cell membrane of its targets (microbes, damaged cells) leading to cell lysis and death. The anaphylatoxins C3a and C5a work to increase vascular permeability, initiate degranulation of mast cells and neutrophils, induce cytokine release from

Complement factor H (CFH) inhibits C3b through complement factor I (CFI) binding. Clusterin and vitronection (Vn) inhibits MAC formation by binding with a complex of C5b-

Covalent bonding of drug molecules to poly(ethylene glycol) (PEG), referred to as PEGylation, is a popular approach to modify and enhance the water solubility and pharmacokinetic and pharmacodynamic properties of biological and small-molecule drugs. In general, PEGs are inert water-soluble polymers, but recently it has been reported that a subretinal injection of PEG induced CNV with dose-dependency via complement activation in mice (Lyzogubov et al., 2011). PEGs can be attached to proteins and other therapeutic molecules, leading to increase the hydrodynamic volume of the therapeutic molecules. In addition, PEGs can shield drugs from interactions with enzymes and from inactivation by the immune system. As a result, PEGylated drugs can exhibit prolonged half-life, higher stability, increased water solubility, and reduced immunogenicity. It is thought that conjugates bearing branched chain PEG show increased thermal stability and higher resistance to enzymatic degradation compared to bearing linear PEG (Hamidi et al., 2008).

Pegaptanib sodium is a chemically-modified oligonucleotide of 28 nucleotides which linked with 40 kDa branched PEG (two arms of 20 kDa linear PEG units), which binds to VEGF165. Pegaptanib was approved by FDA in 2004, and was both the first approved aptamer-based drug and the first approved pharmacotherapy for wet AMD. In rabbit eyes at 24 hours after an intravitreal injection, radiolabeled pegaptanib could be penetrated and distributed in the retina (Eyetech Inc.). In a monkey pharmacokinetics study, pegaptanib was eliminated from the vitreous with a half-life of 94 hours (Drolet et al., 2000), which has been increased by 3.91

work as a C5 convertases.

macrophages, and mediate leukocyte chemotaxis.

convertase).

7.

**4. PEGylation** 

**4.1 Macugen®**

ARC1905 (Ophthotech Corp., Princeton, NJ, U.S.) is a chemically-modified oligonucleotide of 39 nucleotides bound to branched PEG (two arms of 20 kDa linear PEG units), and binds to complement factor C5, leading to prevent the formation of key terminal fragments C5a and MAC (C5b-9). C5a is an important inflammatory activator inducing vascular permeability, recruitment and activation of phagocytes. MAC is involved to initiate cell lysis. Therefore, by inhibiting these C5-mediated inflammation and RPE death leading to geographic atrophy (GA), ARC1905 might be promising for both wet and dry AMD (Kuno & Fujii, 2011a). A phase I study to evaluate the safety, tolerability, and pharmacokinetic profile of multiple doses of intravitreal ARC1905 in combination with multiple doses of Lucentis® is currently in progress. In addition, it has been demonstrated by histopathological examination human dry AMD lesions strongly stained for C5a and MAC at key pathology sites (Anderson et al., 2002). A Phase I clinical trial to evaluate of an intravitreal ARC1905 in patients with GA is undergoing (ClinicalTrials.gov. NCT00950638).

#### **4.3 E10030**

E10030 (Ophthotech Corp.) is a chemically-modified oligonucleotide of 29 nucleotides linked with branched PEG (two arms of 20 kDa linear PEG units), and binds to PDGF-B, which is known to play a role of in the recruitment and maturation of pericytes that can increase resistance to the anti-VEGF treatment for wet AMD. PDGF and its receptor (PDGFR) do not act on vascular endothelial cells, but on pericytes. Therefore, inhibition of PDGF signalling might cause to achieve regression of neovascular vessels. Jo et al. have demonstrated that a combination therapy with anti-VEGF aptamer (Pegaptanib sodium) and anti-PDGFR-β antibody is more effective for CNV prevention and regression compared to monotherapy in the laser-induced CNV model (Jo et al., 2006). In an open-label Phase I clinical study conducted by Ophthotech, 59% of patients treated with E10030 and Lucentis® gained significant vision (3-line gain or better) at 12 weeks after the start of therapy. Interestingly, there was a mean decrease of 86% in the area of CNV at 12 weeks (Ophthotech Corporation). A randomized, controlled, Phase II study of E10030 in combination with Lucentis® for the treatment of wet AMD is currently underway (ClinicalTrials.gov. NCT01089517).

#### **5. Sustained-release systems**

To reduce the frequency of administration, many controlled drug delivery systems have been investigated by using biodegradable or non-biodegradable polymeric devices for the treatment of various retinal diseases as well as AMD (Kuno & Fujii, 2010). In general, drug release from biodegradable matrices consisting of poly(lactide-co-glycolide) (PLGA) is

Clinical Application of Drug Delivery Systems for Treating AMD 235

Despite the remarkable effectiveness for treating wet AMD and other retinal diseases by Lucentis®, patients and physicians have been hoping for an alternative to the frequent intravitreal injections. SurModics, Inc. (Eden Prairie, MN, U.S.) and Genentech, Inc. have been developing a biodegradable microparticles incorporated ranibizumab currently under preclinical stage (SurModics Inc). It is hoped that ranibizumab-loaded microparticles can

Neuroprotective effect of ciliary neurotrophic factor (CNTF) has been confirmed in various animal models of retinal degeneration including light-damaged rats, mutant rhodopsin transgenic mice, and a dog model. In addition, the long-term effect of CNTF has been shown by repeated intravitreal injections of CNTF in an autosomal dominant feline model of rodcone dystrophy or an intravitreal injection of adeno-associated viral (AAV) vectors incorporated CNTF-cDNA in mutant rhodopsin transgenic rats. Neurotech Pharmaceuticals, Inc. (Lincoln, RI, U.S.) has been developing "Encapsulated Cell Technology", which provides an extracellular delivery of CNTF through long-term and stable intraocular release at constant doses through a device implanted in the vitreous. It contains human RPE cell line (ARPE-19) genetically modified to secrete recombinant human CNTF. The device (NT-501) consists of a sealed semi-permeable membrane capsule surrounding a scaffold of 6 strands of polyethylene terephthalate yarn, which can be loaded with cells (length; 6 mm, diameter; 1 mm). The device is surgically implanted in the vitreous through a tiny scleral incision and is anchored by a single suture through a titanium loop at one end of the device. The semi-permeable membrane allows the outward diffusion of CNTF and other cellular metabolites and the inward diffusion of nutrients necessary to support the cell survival in the vitreous cavity while protecting the contents from host cellular immunologic attack.

A Phase I clinical trial for RP has been completed and demonstrated well tolerated for 6 months implantation (Sieving et al., 2006). Eighteen-month results in a Phase II study for patients with GA with dry AMD were reported (Jaffe et al., 2010) (Zhang et al., 2011); participants were randomized in a 2:1:1 ratio to receive a high (20 ng/day) or low dose (5 ng/day) NT-501, or to sham surgery, respectively. Among eyes with baseline best corrected visual acuity (BCVA) 20/63, the mean BCVA in the high dose group was 10.5 and 10.0 letters greater than the low dose/sham group at 12 months (p=0.03) and 18 months, respectively. Stabilized visual acuity was accompanied by the corresponding structural changes; NT-501 treatment resulted in a dose-dependent increase of retinal thickness as early as 4 months after implantation and this increase was maintained through 6, 12 and 18 months (p<0.001). The growth rate of GA area was reduced in treated eyes compared to fellow eyes at 12 and 18 months. In addition, NT-501 also prevented secondary cone

Brimonidine is an α2 adrenergic agonist, which can release various neurotrophins including BDNF, CNTF (Lonngren et al., 2006, Kim et al., 2007), and b-FGF (Lai et al., 2002). These neurotrophins have potential to prevent apoptosis of photoreceptors and/or RPE (Azadi et

deliver ranibizumab over a period of approximately 4 to 6 months (Helzner, 2010).

**5.2 Ranibizumab-loaded microspheres** 

degeneration in RP patients (Talcott et al., 2011).

**5.4 Brimonidine-loaded intravitreal implant** 

**5.3 NT-501** 

degradation-controlled, in contrast, diffusion-controlled drug release is obtained from nonbiodegradable matrices such as silicone and ethylene-vinyl acetate copolymers (EVA). Some sustained-release formulations with constant drug release properties are currently under late clinical stage, but stimuli-responsive formulations with drug release triggered by pathophysiological condition do not exist in developmental stage yet.

#### **5.1 Iluvien®**

It has been reported that activated microglia was accumulated in the degenerative retinas including light-damage mouse (Zhang et al., 2005), rd mouse (Zeiss & Johnson, 2004), Royal College of Surgeons (RCS) rat (Thanos, 1992, Roque et al., 1996), and human eyes of RP and AMD (Gupta et al., 2003). Activated microglia is mainly accumulated within outer nuclear layer and adjacent to the RPE. In contrast, resting microglia shows a downregulated phenotype and a low level of membrane receptors expression; however, it quickly transforms into phagocyte when stimulated by infectious agents, cellular debris, and membrane fragments, such as lipopolysaccharides (Kreutzberg, 1996, Gehrmann, 1996, Pawate et al., 2004, Whitton, 2007). Within 24 hours of activation, microglial cells enlarge, acquire an ameboid macrophage-like shape, leading to increased microglial IgG reactivity and upregulation of complement receptors, and intercellular adhesion molecules (Orr et al., 2002). Activated microglia releases cytotoxic molecules, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10, interferon (IFN)-γ, hydrogen peroxide, and superoxide anion (Orr et al., 2002, Boje & Arora, 1992, Banati et al., 1993, Kreutzberg, 1995, Kim & de Vellis, 2005), which may induce apoptosis in otherwise healthy cells such as photoreceptors, RPE, and vascular endothelial cells. Once the activating stimulus is eliminated, microglia quickly returns to their resting state. While the stimulus continues, however, microglial cells express major histocompatibility complex (MHC) class I and II (Kreutzberg, 1995, Nakanishi, 2003) and inflammatory glycoproteins (Aloisi, 2001), which are self-stimulating and stimulate/recruit other immune cells. Microglia then clusters around neurons, adheres to their surfaces, continually produces cytotoxins that leads to neuronal death, and consequently recruit and activate additional microglia (Kreutzberg, 1996, Banati et al., 1993, Klegeris & McGeer, 2000) via chemokines such as CCL-5 (RANTES), macrophage inflammation protein (MIP)-1α and MIP-1β, monocyte chemoattractant protein (MCP)-1 and MCP-3 (Boje & Arora, 1992, Banati et al., 1993, McGeer et al., 1993, Min et al., 2004).

Recently, a retinal neuroprotective effect of sustained-release of a corticosteroid, fluocinolone acetonide (FA) for progressive retinal degeneration has been demonstrated in RCS rat (Glybina et al., 2009) and S334ter mutant rhodopsin transgenic rats (Glybina et al., 2010). In both animal models, FA treatment was associated with significant decrease in the number of microglial cells in both the outer and inner nuclear layer. In addition, corticosteroids have a genomic neuroprotective effect via Trk activation, leading to a trophic effect (Jeanneteau et al., 2008). An injectable, rod-shaped intravitreal implant with FA (Iluvien®; length: 3.5 mm, diameter: 0.37 mm, formerly Medidur™) has been developed by Alimera Sciences (Alpharetta, GA, U.S.) for the treatment of dry AMD under Phase II study (ClinicalTrials.gov. NCT00695318). Furthermore, the feasibility study of Medidur™ as a maintenance therapy for wet AMD patients who have been treated with Lucentis® for at least 6 months and have reached a plateau is currently in a pilot Phase II (ClinicalTrials.gov. NCT00605423).

#### **5.2 Ranibizumab-loaded microspheres**

Despite the remarkable effectiveness for treating wet AMD and other retinal diseases by Lucentis®, patients and physicians have been hoping for an alternative to the frequent intravitreal injections. SurModics, Inc. (Eden Prairie, MN, U.S.) and Genentech, Inc. have been developing a biodegradable microparticles incorporated ranibizumab currently under preclinical stage (SurModics Inc). It is hoped that ranibizumab-loaded microparticles can deliver ranibizumab over a period of approximately 4 to 6 months (Helzner, 2010).

#### **5.3 NT-501**

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

degradation-controlled, in contrast, diffusion-controlled drug release is obtained from nonbiodegradable matrices such as silicone and ethylene-vinyl acetate copolymers (EVA). Some sustained-release formulations with constant drug release properties are currently under late clinical stage, but stimuli-responsive formulations with drug release triggered by

It has been reported that activated microglia was accumulated in the degenerative retinas including light-damage mouse (Zhang et al., 2005), rd mouse (Zeiss & Johnson, 2004), Royal College of Surgeons (RCS) rat (Thanos, 1992, Roque et al., 1996), and human eyes of RP and AMD (Gupta et al., 2003). Activated microglia is mainly accumulated within outer nuclear layer and adjacent to the RPE. In contrast, resting microglia shows a downregulated phenotype and a low level of membrane receptors expression; however, it quickly transforms into phagocyte when stimulated by infectious agents, cellular debris, and membrane fragments, such as lipopolysaccharides (Kreutzberg, 1996, Gehrmann, 1996, Pawate et al., 2004, Whitton, 2007). Within 24 hours of activation, microglial cells enlarge, acquire an ameboid macrophage-like shape, leading to increased microglial IgG reactivity and upregulation of complement receptors, and intercellular adhesion molecules (Orr et al., 2002). Activated microglia releases cytotoxic molecules, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-10, interferon (IFN)-γ, hydrogen peroxide, and superoxide anion (Orr et al., 2002, Boje & Arora, 1992, Banati et al., 1993, Kreutzberg, 1995, Kim & de Vellis, 2005), which may induce apoptosis in otherwise healthy cells such as photoreceptors, RPE, and vascular endothelial cells. Once the activating stimulus is eliminated, microglia quickly returns to their resting state. While the stimulus continues, however, microglial cells express major histocompatibility complex (MHC) class I and II (Kreutzberg, 1995, Nakanishi, 2003) and inflammatory glycoproteins (Aloisi, 2001), which are self-stimulating and stimulate/recruit other immune cells. Microglia then clusters around neurons, adheres to their surfaces, continually produces cytotoxins that leads to neuronal death, and consequently recruit and activate additional microglia (Kreutzberg, 1996, Banati et al., 1993, Klegeris & McGeer, 2000) via chemokines such as CCL-5 (RANTES), macrophage inflammation protein (MIP)-1α and MIP-1β, monocyte chemoattractant protein (MCP)-1 and

MCP-3 (Boje & Arora, 1992, Banati et al., 1993, McGeer et al., 1993, Min et al., 2004).

Recently, a retinal neuroprotective effect of sustained-release of a corticosteroid, fluocinolone acetonide (FA) for progressive retinal degeneration has been demonstrated in RCS rat (Glybina et al., 2009) and S334ter mutant rhodopsin transgenic rats (Glybina et al., 2010). In both animal models, FA treatment was associated with significant decrease in the number of microglial cells in both the outer and inner nuclear layer. In addition, corticosteroids have a genomic neuroprotective effect via Trk activation, leading to a trophic effect (Jeanneteau et al., 2008). An injectable, rod-shaped intravitreal implant with FA (Iluvien®; length: 3.5 mm, diameter: 0.37 mm, formerly Medidur™) has been developed by Alimera Sciences (Alpharetta, GA, U.S.) for the treatment of dry AMD under Phase II study (ClinicalTrials.gov. NCT00695318). Furthermore, the feasibility study of Medidur™ as a maintenance therapy for wet AMD patients who have been treated with Lucentis® for at least 6 months and have reached a plateau is currently in a pilot Phase II (ClinicalTrials.gov.

pathophysiological condition do not exist in developmental stage yet.

**5.1 Iluvien®**

NCT00605423).

Neuroprotective effect of ciliary neurotrophic factor (CNTF) has been confirmed in various animal models of retinal degeneration including light-damaged rats, mutant rhodopsin transgenic mice, and a dog model. In addition, the long-term effect of CNTF has been shown by repeated intravitreal injections of CNTF in an autosomal dominant feline model of rodcone dystrophy or an intravitreal injection of adeno-associated viral (AAV) vectors incorporated CNTF-cDNA in mutant rhodopsin transgenic rats. Neurotech Pharmaceuticals, Inc. (Lincoln, RI, U.S.) has been developing "Encapsulated Cell Technology", which provides an extracellular delivery of CNTF through long-term and stable intraocular release at constant doses through a device implanted in the vitreous. It contains human RPE cell line (ARPE-19) genetically modified to secrete recombinant human CNTF. The device (NT-501) consists of a sealed semi-permeable membrane capsule surrounding a scaffold of 6 strands of polyethylene terephthalate yarn, which can be loaded with cells (length; 6 mm, diameter; 1 mm). The device is surgically implanted in the vitreous through a tiny scleral incision and is anchored by a single suture through a titanium loop at one end of the device. The semi-permeable membrane allows the outward diffusion of CNTF and other cellular metabolites and the inward diffusion of nutrients necessary to support the cell survival in the vitreous cavity while protecting the contents from host cellular immunologic attack.

A Phase I clinical trial for RP has been completed and demonstrated well tolerated for 6 months implantation (Sieving et al., 2006). Eighteen-month results in a Phase II study for patients with GA with dry AMD were reported (Jaffe et al., 2010) (Zhang et al., 2011); participants were randomized in a 2:1:1 ratio to receive a high (20 ng/day) or low dose (5 ng/day) NT-501, or to sham surgery, respectively. Among eyes with baseline best corrected visual acuity (BCVA) 20/63, the mean BCVA in the high dose group was 10.5 and 10.0 letters greater than the low dose/sham group at 12 months (p=0.03) and 18 months, respectively. Stabilized visual acuity was accompanied by the corresponding structural changes; NT-501 treatment resulted in a dose-dependent increase of retinal thickness as early as 4 months after implantation and this increase was maintained through 6, 12 and 18 months (p<0.001). The growth rate of GA area was reduced in treated eyes compared to fellow eyes at 12 and 18 months. In addition, NT-501 also prevented secondary cone degeneration in RP patients (Talcott et al., 2011).

#### **5.4 Brimonidine-loaded intravitreal implant**

Brimonidine is an α2 adrenergic agonist, which can release various neurotrophins including BDNF, CNTF (Lonngren et al., 2006, Kim et al., 2007), and b-FGF (Lai et al., 2002). These neurotrophins have potential to prevent apoptosis of photoreceptors and/or RPE (Azadi et

Clinical Application of Drug Delivery Systems for Treating AMD 237

Steba Biotech S.A. currently conducts a Phase II study for WST-11 PDT in wet AMD patients

Tissue factor (TF) acts as a primary cellular initiator of blood coagulation, and has following additional biological functions involving neovascularization. TF can induce angiogenesis by upregulating VEGF and also promote angiogenesis via TF-initiated coagulation pathways. Thrombin stimulation of platelets, which is a major VEGF transporter, releases VEGF (Mohle et al., 1997), leading to stimulate endothelial cells to induce and expose more TF, following further thrombin formation. In addition, TF expressed in surgically excited CNV membrane and AMD eyes was related to active inflammation site accompanied by an accumulation of macrophages and fibrin deposition (Grossniklaus et al., 2002). It has been reported that TF mRNA expression in AMD was 32-fold higher than in the non-AMD (Cho et al., 2011) and TF was expressed only on neovascular endothelial cells not normal vascular endothelial cells (Contrino et al., 1996). Therefore, TF might be a specific target for

hI-con1 (Iconic Therapeutics, Inc., Atlanta, GA, U.S.) is a chimeric IgG-like homodimeric protein composed of a targeting-domain (mutated, inactivated factor VIIa, which is a ligand for TF) fused to an effector-domain (human IgG Fc) with an intact hinge region (Iconic Therapeutics). Once hI-con1 binds to TF on the surface of neovascular endothelial cells, the effector-domain mobilizes natural killer (NK) cells mediated via the Fc receptor, leading to activating the complement cascade (Wang et al., 1999, Hu & Li, 2010) and inducing the selective apoptosis of TF-expressing cells. Consequently, NK cells do not induce apoptosis of other cells including normal vascular tissue and RPE and neural retina. Bora et al. have demonstrated that intravitreal mouse factor VII-human IgG1 Fc chimeric conjugate inhibited CNV in a laser-induced CNV model in mice (Bora et al., 2003). In addition, Tezel et al. reported that this immunoprotein could selectively regress already-established CNV in laser-induced pig model (Tezel et al., 2007). A Phase I/IIa study of intravitreal hI-con1 for

VEGFR2 (Flk-1) plays a pivotal role in endothelial cell proliferation and migration (Millauer et al., 1993, Risau, 1997), and is upregulated during CNV formation (Wada et al., 1999). VEGFR2 vaccination therapy has been progressed in the cancer field (Niethammer et al., 2002, Wada et al., 2005, Pan et al., 2008). The strategy of VEGFR vaccination therapy for wet AMD is to induce apoptosis of neovascular endothelial cells, and inhibition and regression of CNV by cytotoxic T lymphocytes (CTLs). Takahashi et al. have demonstrated that vaccination with human VEGFR2-derived epitope peptide (VEGFR2-773) significantly inhibited CNV in laser-induced A2/Kb transgenic mice, which express chimeric humanmouse MHC class I molecule, and this chimeric molecule shows 71% concordance with the human CTL repertoire (Vitiello et al., 1991). VEGFR2 peptide induces CTLs in the histocompatibility leukocyte antigen (HLA) class I-restricted manner (Wada et al., 2005).

It is thought that the advantage of VEGFR2 vaccination is long-lasting therapeutic effect on the vascular endothelial cells since endothelial cells are genetically stable and do not show

(ClinicalTrials.gov. NCT01021956).

**6.3 I-con1** 

neovascular tissues.

**6.4 Anti-VEGFR vaccine** 

wet AMD is currently underway (Iconic Therapeutics).

al., 2007, Zhang et al., 2009). A biodegradable, rod-shaped PLGA intravitreal implant containing brimonidine tartrate is now in a Phase II clinical study for dry AMD (ClinicalTrials.gov. NCT00658619) by Allergan Inc. (Irvine, CA, U.S.).

#### **6. Targeting systems**

Selective targeting to the neovascular lesions is desired for the improvement of therapeutic efficacy and the reduction of normal tissue damage. Active targeting to neovascular endothelial cells using highly-expressed specific molecules on endothelial cells has been widely investigated for the treatment of CNV. In addition, immunotherapy in conjunction with active targeting is also developing for regression of CNV.

#### **6.1 Visudyne®**

Visudyne® (QLT Ophthalmics, Inc., Menlo Park, CA, U.S.) is an intravenous liposomal formulation containing a photosensitizer, verteporfin in PDT for predominantly classic subfoveal CNV due to wet AMD, pathologic myopia or presumed ocular histoplasmosis (QLT Ophthalmics). Plasma lipoproteins, such as low-density lipoprotein (LDL), have been proposed to enhance the delivery of hydrophobic verteporfin to malignant tissue since tumor cells have increased the number of LDL receptors (Allison et al., 1994). In addition, liposomes composed of negatively charged phospholipids such as phosphatidylglycerol are taken up into tumor cells by LDL receptor-mediated endocytosis (Amin et al., 2002). It is thought that verteporfin released into the blood stream from liposomes is associated with LDL and is taken up into neovascular tissue, on the other hand, un-dissociated verteporfin, which is still encapsulated in the liposomes, is selectively accumulated in neovascular endothelial cells via LDL receptor-mediated endocytosis, since phosphatidylglycerol is the major constituent of Visudyne®.

Since LDL receptors are also expressed in RPE as well as endothelial cells (Hayes et al., 1989), verteporfin PDT causes damage to RPE associated with photoreceptor lesions. Indeed, adverse effects by verteporfin PDT have been reported (Tzekov et al., 2006, Ozdemir et al., 2006, Oner et al., 2005a, 2005b) in clinical situation. To enhance PDT effects and minimize damage of normal tissues, highly selective targeting might be necessary.

#### **6.2 WST-11 (Stakel® )**

Serum albumin has the unique ability to reversibly or covalently bind various endogenous or exogenous ligands with high affinity, resulting in working as a transporter and depot protein for various compounds (Kragh-Hansen, 1990). The cellular uptake of serum albumin via receptor (albondin)-mediated endocytosis (Schnitzer & Oh, 1994, John et al., 2001) might cause highly efficient intracellular trafficking. WST-11 (Stakel®, Steba Biotech S.A., Toussus-Le-Noble, France) is a negatively charged, water-soluble bacteriochlorophyll derivative with maximum absorption wavelength in the near infrared (753 nm) and rapid clearance from the body (Mazor et al., 2005, Brandis et al., 2005). WST-11 binds to serum albumin and has potent anti-neovascularization via the generation of hydroxyl radicals when stimulated by the proper light wavelength. Berdugo et al. have demonstrated that WST-11 PDT, which selectively occludes CNV, could be achieved in laser-induced CNV model of rats without the damages to the retinal tissues such as RPE and photoreceptors unlike verteporfin PDT. Steba Biotech S.A. currently conducts a Phase II study for WST-11 PDT in wet AMD patients (ClinicalTrials.gov. NCT01021956).

#### **6.3 I-con1**

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

al., 2007, Zhang et al., 2009). A biodegradable, rod-shaped PLGA intravitreal implant containing brimonidine tartrate is now in a Phase II clinical study for dry AMD

Selective targeting to the neovascular lesions is desired for the improvement of therapeutic efficacy and the reduction of normal tissue damage. Active targeting to neovascular endothelial cells using highly-expressed specific molecules on endothelial cells has been widely investigated for the treatment of CNV. In addition, immunotherapy in conjunction

Visudyne® (QLT Ophthalmics, Inc., Menlo Park, CA, U.S.) is an intravenous liposomal formulation containing a photosensitizer, verteporfin in PDT for predominantly classic subfoveal CNV due to wet AMD, pathologic myopia or presumed ocular histoplasmosis (QLT Ophthalmics). Plasma lipoproteins, such as low-density lipoprotein (LDL), have been proposed to enhance the delivery of hydrophobic verteporfin to malignant tissue since tumor cells have increased the number of LDL receptors (Allison et al., 1994). In addition, liposomes composed of negatively charged phospholipids such as phosphatidylglycerol are taken up into tumor cells by LDL receptor-mediated endocytosis (Amin et al., 2002). It is thought that verteporfin released into the blood stream from liposomes is associated with LDL and is taken up into neovascular tissue, on the other hand, un-dissociated verteporfin, which is still encapsulated in the liposomes, is selectively accumulated in neovascular endothelial cells via LDL receptor-mediated endocytosis, since phosphatidylglycerol is the

Since LDL receptors are also expressed in RPE as well as endothelial cells (Hayes et al., 1989), verteporfin PDT causes damage to RPE associated with photoreceptor lesions. Indeed, adverse effects by verteporfin PDT have been reported (Tzekov et al., 2006, Ozdemir et al., 2006, Oner et al., 2005a, 2005b) in clinical situation. To enhance PDT effects and

Serum albumin has the unique ability to reversibly or covalently bind various endogenous or exogenous ligands with high affinity, resulting in working as a transporter and depot protein for various compounds (Kragh-Hansen, 1990). The cellular uptake of serum albumin via receptor (albondin)-mediated endocytosis (Schnitzer & Oh, 1994, John et al., 2001) might cause highly efficient intracellular trafficking. WST-11 (Stakel®, Steba Biotech S.A., Toussus-Le-Noble, France) is a negatively charged, water-soluble bacteriochlorophyll derivative with maximum absorption wavelength in the near infrared (753 nm) and rapid clearance from the body (Mazor et al., 2005, Brandis et al., 2005). WST-11 binds to serum albumin and has potent anti-neovascularization via the generation of hydroxyl radicals when stimulated by the proper light wavelength. Berdugo et al. have demonstrated that WST-11 PDT, which selectively occludes CNV, could be achieved in laser-induced CNV model of rats without the damages to the retinal tissues such as RPE and photoreceptors unlike verteporfin PDT.

minimize damage of normal tissues, highly selective targeting might be necessary.

(ClinicalTrials.gov. NCT00658619) by Allergan Inc. (Irvine, CA, U.S.).

with active targeting is also developing for regression of CNV.

**6. Targeting systems** 

major constituent of Visudyne®.

**)** 

**6.2 WST-11 (Stakel®**

**6.1 Visudyne®**

Tissue factor (TF) acts as a primary cellular initiator of blood coagulation, and has following additional biological functions involving neovascularization. TF can induce angiogenesis by upregulating VEGF and also promote angiogenesis via TF-initiated coagulation pathways. Thrombin stimulation of platelets, which is a major VEGF transporter, releases VEGF (Mohle et al., 1997), leading to stimulate endothelial cells to induce and expose more TF, following further thrombin formation. In addition, TF expressed in surgically excited CNV membrane and AMD eyes was related to active inflammation site accompanied by an accumulation of macrophages and fibrin deposition (Grossniklaus et al., 2002). It has been reported that TF mRNA expression in AMD was 32-fold higher than in the non-AMD (Cho et al., 2011) and TF was expressed only on neovascular endothelial cells not normal vascular endothelial cells (Contrino et al., 1996). Therefore, TF might be a specific target for neovascular tissues.

hI-con1 (Iconic Therapeutics, Inc., Atlanta, GA, U.S.) is a chimeric IgG-like homodimeric protein composed of a targeting-domain (mutated, inactivated factor VIIa, which is a ligand for TF) fused to an effector-domain (human IgG Fc) with an intact hinge region (Iconic Therapeutics). Once hI-con1 binds to TF on the surface of neovascular endothelial cells, the effector-domain mobilizes natural killer (NK) cells mediated via the Fc receptor, leading to activating the complement cascade (Wang et al., 1999, Hu & Li, 2010) and inducing the selective apoptosis of TF-expressing cells. Consequently, NK cells do not induce apoptosis of other cells including normal vascular tissue and RPE and neural retina. Bora et al. have demonstrated that intravitreal mouse factor VII-human IgG1 Fc chimeric conjugate inhibited CNV in a laser-induced CNV model in mice (Bora et al., 2003). In addition, Tezel et al. reported that this immunoprotein could selectively regress already-established CNV in laser-induced pig model (Tezel et al., 2007). A Phase I/IIa study of intravitreal hI-con1 for wet AMD is currently underway (Iconic Therapeutics).

#### **6.4 Anti-VEGFR vaccine**

VEGFR2 (Flk-1) plays a pivotal role in endothelial cell proliferation and migration (Millauer et al., 1993, Risau, 1997), and is upregulated during CNV formation (Wada et al., 1999). VEGFR2 vaccination therapy has been progressed in the cancer field (Niethammer et al., 2002, Wada et al., 2005, Pan et al., 2008). The strategy of VEGFR vaccination therapy for wet AMD is to induce apoptosis of neovascular endothelial cells, and inhibition and regression of CNV by cytotoxic T lymphocytes (CTLs). Takahashi et al. have demonstrated that vaccination with human VEGFR2-derived epitope peptide (VEGFR2-773) significantly inhibited CNV in laser-induced A2/Kb transgenic mice, which express chimeric humanmouse MHC class I molecule, and this chimeric molecule shows 71% concordance with the human CTL repertoire (Vitiello et al., 1991). VEGFR2 peptide induces CTLs in the histocompatibility leukocyte antigen (HLA) class I-restricted manner (Wada et al., 2005).

It is thought that the advantage of VEGFR2 vaccination is long-lasting therapeutic effect on the vascular endothelial cells since endothelial cells are genetically stable and do not show

Clinical Application of Drug Delivery Systems for Treating AMD 239

RetinoStat® (Oxford Biomedica, Oxford, UK) is an equine infectious anaemia virus (EIAV) based lentiviral vector expressing human endostatin and angiostatin, which are endogenous angiostatic factors. EIAV can transduce both dividing and non-dividing cells. Endostatin is an internal fragment of collagen XVIII, and downregulates the expression of the antiapoptotic proteins such as Bcl-2 and Bcl-XL (Dhanabal et al., 1999), and may interact with endothelial cell surface receptors and integrins leading to apoptosis of endothelial cells in active neovascularization, but not mature vasculature. In addition, endostatin blocks VEGF signalling via a direct interaction with VEGFR2 (Kim et al., 2002). Angiostatin is a cleavage product of plasminogen, which has the kringle domains, and promotes apoptosis of proliferating vascular endothelial cells similar to endostatin (Claesson-Welsh et al., 1998, Hari et al., 2000). Also, angiostatin downregulates VEGF expression (Hajitou et al., 2002, Sima et al., 2004). RetinoStat® incorporates RPE-specific vitelliform macular dystrophy gene (VMD2) promoter, leading to limited transgene expression to RPE after a subretinal injection (Kan et al., 2009). Kachi et al. have demonstrated that a subretinal injection of RetinoStat® significantly inhibited CNV in laser-induced CNV model of mice (Kachi et al., 2009). A Phase I study of subretinal RetinoStat® in wet AMD patients is currently ongoing

Mechanical devices have been developing for the purpose of more selective drug targeting,

A microcatheter, iTrack™ 250A (iScience Interventional™, Menlo Park, CA, U.S.) is originally designed for canaloplasty (iScience Interventional™), which is a new treatment for glaucoma (Lewis et al., 2009, 2011). The iTrack™ 250A consists of an optical fiber to allow transmission of light to the microcannula tip for surgical illumination and guidance. Recently, this microcatheter is challenged to use for suprachoroidal drug delivery (Olsen, 2007, Rizzo et al., 2010). The pharmacokinetic study of suprachoroidal delivery of TA in pigs has shown that TA remained in the ocular tissues for at least 120 days, and the systemic exposure was very low (Olsen et al., 2006). In contrast, the study to compare the pharmacokinetics of bevacizumab between intravitreal and suprachoroidal injections to pigs (Olsen et al., 2011) reported that the profile of intravitreal injections of bevacizumab was more sustained than that of suprachoroidal injections at the same dosage level. Intravitreal injected bevacizumab distributed more to the inner retina, whereas suprachoroidal injected bevacizumab distributed primarily to the choroid, RPE, and photoreceptor outer segments. Scharioth et al. have tried to conduct suprachoroidal injections of bevacizumab in the wet AMD patients who were non-responder of intravitreal anti-VEGF therapy and/or initial BCVA < 0.05 (Scharioth et al., 2011). In the case of the patient who had a history of 21 intravitreal anti-VEGF injections with poor response to this therapy, and BCVA was 0.1, a significant reduction of pigment epithelial detachment was observed at 4 weeks after a suprachoroidal injection of bevacizumab, BCVA slightly improved to 0.16 and the subfoveal

**7.3 RetinoStat®**

(ClinicalTrials.gov. NCT01301443).

chronic infusion, or stimuli-responsive drug release.

**8. Devices** 

**8.1 Microcatheter** 

the downregulation of HLA class I molecules (Niethammer et al., 2002). It has been reported that, 60.8% and 19.9% in Japanese population share a common HLA-A\*2402 allele and HLA-A\*0201 allele, respectively (Date et al., 1996). HLA-A\*2402 restricted VEGFR1- and VEGFR2 derived peptide vaccination therapy for wet AMD is conducted under a Phase I study in Japan (ClinicalTrials.gov. NCT00791570).

#### **7. Gene therapy**

Adenoviral (Ad) and AAV vectors are non-integrating and transduce both dividing and non-dividing cells. However, Ad and AAV elicit CTLs-mediated immune responses resulting in limitation of duration of transgene expression (McConnell & Imperiale, 2004). Helper-dependent Ad can extend the duration of ocular expression from less than 3 months to up to 1 year (Lamartina et al., 2007). Lentiviral vectors can induce stable, long-term transgene expression in the retinal (Balaggan et al., 2006). Lentivirus, which are integrating vectors, have the risk of insertional oncogenesis. Highly deleted (Molina et al., 2004), selfinactivating (Berkowitz et al., 2001) and non-integrating (Yanez-Munoz et al., 2006) lentiviral vectors have been developed as safer vectors. In general, subretinal injections are conducted in the operating room and are more invasive than intravitreal injections. If a single subretinal injection of a vector to provide prolonged suppression of CNV, it might be reasonable and feasible to substitute for repeated intravitreal injections of Lucentis®.

#### **7.1 Pigment epithelium-derived factor**

AdPEDF.11D is E1-, partial E3-, and E4-deleted Ad vector, which is replication-deficient, expressing pigment epithelium-derived factor (PEDF). A Phase I study of intravitreal AdPEDF.11D conducted by GenVec, Inc. (Gaithersburg, MD, U.S.) has completed. The results have shown that several complications such as mild inflammation, corneal edema, and elevated IOP were observed in some patients, but systemic hematogenous vector spread and systemic immune responses were not observed. Although hyperpermeability appeared to resolve in some patients received high-dose AdPEDF.11D, unfortunately, patients received high-dose (108-109.5 particle units) had no change in visual acuity compared to low-dose (106-107.5 particle units) patients whose visual acuity appeared to worsen over the course of study (Campochiaro et al., 2006). Further clinical trials have not progressed after the completion of a Phase I study in 2006.

#### **7.2 sFLT01**

sFLT01 is an antiangiogenic fusion protein consisting of the VEGF/placental growth factor (PIGF) binding domain of human Flt-1 (hVEGFR1) fused to the Fc portion of human IgG1 through a polyglycine linker (Bagley et al., 2011). Therefore, sFLT01 acts as a VEGF decoy. It has been reported that an intravitreal injection of AAV serotype 2 (AAV2) vector coding for sFLT01 (AAV2-sFLT01) significantly inhibited CNV in laser-induced CNV model of mice and monkeys (Lukason et al., 2011) and retinal neovascularization in mouse oxygeninduced retinopathy model (Pechan et al., 2009). Interestingly, sFLT01 expression in the retina continued for up to 12 months after an intravitreal injection (Pechan et al., 2009). A Phase I clinical trial of AAV2-sFLT01 is currently conducted by Genzyme (Cambridge, MA, U.S.) (ClinicalTrials.gov. NCT01024998).

#### **7.3 RetinoStat®**

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

the downregulation of HLA class I molecules (Niethammer et al., 2002). It has been reported that, 60.8% and 19.9% in Japanese population share a common HLA-A\*2402 allele and HLA-A\*0201 allele, respectively (Date et al., 1996). HLA-A\*2402 restricted VEGFR1- and VEGFR2 derived peptide vaccination therapy for wet AMD is conducted under a Phase I study in

Adenoviral (Ad) and AAV vectors are non-integrating and transduce both dividing and non-dividing cells. However, Ad and AAV elicit CTLs-mediated immune responses resulting in limitation of duration of transgene expression (McConnell & Imperiale, 2004). Helper-dependent Ad can extend the duration of ocular expression from less than 3 months to up to 1 year (Lamartina et al., 2007). Lentiviral vectors can induce stable, long-term transgene expression in the retinal (Balaggan et al., 2006). Lentivirus, which are integrating vectors, have the risk of insertional oncogenesis. Highly deleted (Molina et al., 2004), selfinactivating (Berkowitz et al., 2001) and non-integrating (Yanez-Munoz et al., 2006) lentiviral vectors have been developed as safer vectors. In general, subretinal injections are conducted in the operating room and are more invasive than intravitreal injections. If a single subretinal injection of a vector to provide prolonged suppression of CNV, it might be

reasonable and feasible to substitute for repeated intravitreal injections of Lucentis®.

AdPEDF.11D is E1-, partial E3-, and E4-deleted Ad vector, which is replication-deficient, expressing pigment epithelium-derived factor (PEDF). A Phase I study of intravitreal AdPEDF.11D conducted by GenVec, Inc. (Gaithersburg, MD, U.S.) has completed. The results have shown that several complications such as mild inflammation, corneal edema, and elevated IOP were observed in some patients, but systemic hematogenous vector spread and systemic immune responses were not observed. Although hyperpermeability appeared to resolve in some patients received high-dose AdPEDF.11D, unfortunately, patients received high-dose (108-109.5 particle units) had no change in visual acuity compared to low-dose (106-107.5 particle units) patients whose visual acuity appeared to worsen over the course of study (Campochiaro et al., 2006). Further clinical trials have not

sFLT01 is an antiangiogenic fusion protein consisting of the VEGF/placental growth factor (PIGF) binding domain of human Flt-1 (hVEGFR1) fused to the Fc portion of human IgG1 through a polyglycine linker (Bagley et al., 2011). Therefore, sFLT01 acts as a VEGF decoy. It has been reported that an intravitreal injection of AAV serotype 2 (AAV2) vector coding for sFLT01 (AAV2-sFLT01) significantly inhibited CNV in laser-induced CNV model of mice and monkeys (Lukason et al., 2011) and retinal neovascularization in mouse oxygeninduced retinopathy model (Pechan et al., 2009). Interestingly, sFLT01 expression in the retina continued for up to 12 months after an intravitreal injection (Pechan et al., 2009). A Phase I clinical trial of AAV2-sFLT01 is currently conducted by Genzyme (Cambridge, MA,

Japan (ClinicalTrials.gov. NCT00791570).

**7.1 Pigment epithelium-derived factor** 

U.S.) (ClinicalTrials.gov. NCT01024998).

progressed after the completion of a Phase I study in 2006.

**7. Gene therapy** 

**7.2 sFLT01** 

RetinoStat® (Oxford Biomedica, Oxford, UK) is an equine infectious anaemia virus (EIAV) based lentiviral vector expressing human endostatin and angiostatin, which are endogenous angiostatic factors. EIAV can transduce both dividing and non-dividing cells. Endostatin is an internal fragment of collagen XVIII, and downregulates the expression of the antiapoptotic proteins such as Bcl-2 and Bcl-XL (Dhanabal et al., 1999), and may interact with endothelial cell surface receptors and integrins leading to apoptosis of endothelial cells in active neovascularization, but not mature vasculature. In addition, endostatin blocks VEGF signalling via a direct interaction with VEGFR2 (Kim et al., 2002). Angiostatin is a cleavage product of plasminogen, which has the kringle domains, and promotes apoptosis of proliferating vascular endothelial cells similar to endostatin (Claesson-Welsh et al., 1998, Hari et al., 2000). Also, angiostatin downregulates VEGF expression (Hajitou et al., 2002, Sima et al., 2004). RetinoStat® incorporates RPE-specific vitelliform macular dystrophy gene (VMD2) promoter, leading to limited transgene expression to RPE after a subretinal injection (Kan et al., 2009). Kachi et al. have demonstrated that a subretinal injection of RetinoStat® significantly inhibited CNV in laser-induced CNV model of mice (Kachi et al., 2009). A Phase I study of subretinal RetinoStat® in wet AMD patients is currently ongoing (ClinicalTrials.gov. NCT01301443).

#### **8. Devices**

Mechanical devices have been developing for the purpose of more selective drug targeting, chronic infusion, or stimuli-responsive drug release.

#### **8.1 Microcatheter**

A microcatheter, iTrack™ 250A (iScience Interventional™, Menlo Park, CA, U.S.) is originally designed for canaloplasty (iScience Interventional™), which is a new treatment for glaucoma (Lewis et al., 2009, 2011). The iTrack™ 250A consists of an optical fiber to allow transmission of light to the microcannula tip for surgical illumination and guidance. Recently, this microcatheter is challenged to use for suprachoroidal drug delivery (Olsen, 2007, Rizzo et al., 2010). The pharmacokinetic study of suprachoroidal delivery of TA in pigs has shown that TA remained in the ocular tissues for at least 120 days, and the systemic exposure was very low (Olsen et al., 2006). In contrast, the study to compare the pharmacokinetics of bevacizumab between intravitreal and suprachoroidal injections to pigs (Olsen et al., 2011) reported that the profile of intravitreal injections of bevacizumab was more sustained than that of suprachoroidal injections at the same dosage level. Intravitreal injected bevacizumab distributed more to the inner retina, whereas suprachoroidal injected bevacizumab distributed primarily to the choroid, RPE, and photoreceptor outer segments. Scharioth et al. have tried to conduct suprachoroidal injections of bevacizumab in the wet AMD patients who were non-responder of intravitreal anti-VEGF therapy and/or initial BCVA < 0.05 (Scharioth et al., 2011). In the case of the patient who had a history of 21 intravitreal anti-VEGF injections with poor response to this therapy, and BCVA was 0.1, a significant reduction of pigment epithelial detachment was observed at 4 weeks after a suprachoroidal injection of bevacizumab, BCVA slightly improved to 0.16 and the subfoveal

Clinical Application of Drug Delivery Systems for Treating AMD 241

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Bibbiani, F., Oh, J. D. & Chase, T. N. (2001). Serotonin 5-HT1A agonist improves motor

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progressive courses of the targeted disease.

**10. References** 

833-839

No.3, 411-431

Vol.8, No.3, 275-285

No.10, 1829-1834

membrane totally disappeared at 8 weeks. During 6 months of follow-up, no signs of recurrence were observed.

#### **8.2 Micropump™ system**

A microelectromechanical systems (MEMS) drug delivery device is investigated for the treatment of chronic and refractory ocular diseases (Lo et al., 2009, Saati et al., 2010). MEMS device can be re-filled with the drug solution, giving long-term drug therapy which avoids repeated surgeries. The first generation of MEMS is a manually-controlled system limited by variations in the drug-release duration and force applied for depressing of the reservoir. To resolve this problem, the next generation device consists of an electrolysis chamber with electrolysis actuation to precisely delivery the desired dosage volume, a drug reservoir with refill port, battery and electronics. Biocompatible and flexible parylene is used to construct the MEMS. Battery and wireless inductive power transfer can be used to drive electrolysis. Electrolysis is a low power process in which the electrochemically-induced phase change of water to hydrogen and oxygen gas generates pressure in the reservoir forcing the drug through the cannula (Saati et al., 2010). The reservoir is implanted in the subconjunctival space and flexible cannula is inserted through incision into the anterior or posterior segment. Gonzalez-Soto et al. have demonstrated that a slower prolonged infusion of the same volume and concentration of intravitreal ranibizumab is equivalent to a bolus intravitreal injection of ranibizumab to human VEGF-induced retinal hyperpermeability model of rabbits (Gonzalez-Soto et al., 2011).

Replenish, Inc. (Pasadena, CA, U.S.) plans to enter clinical trials for a refillable and programmable pump that would be implanted in the eye to feed medicine for glaucoma or AMD. The Replenish device can last more than 5 years before needing replacement, much longer than current treatments (Flanigan, 2009).

#### **8.3 ODTx**

On Demand Therapeutics, Inc. (Menlo Park, CA, U.S.) has been developing a multi-reservoir implantable device for laser-activated drug delivery to the posterior segment of the eye (RetinaToday, 2010) (On Demand Therapeutics Inc). The injectable, biocompatible, nonresorbable device (ODTx) contains reservoirs designed for drug release in optimized doses. The reservoirs are capable of storing small- or large-molecule drugs that can be released via a standard, non-invasive laser activation procedure. The multiple reservoir system allows for ophthalmologists to control drug delivery by activating specific reservoirs, while unactivated reservoirs remain intact. Unfortunately, clinical trials of ODTx have not progressed yet.

#### **9. Conclusion**

Recent advances in drug delivery systems under clinical situation and in the late experimental stages are described in this chapter. AMD is chronic, progressive and refractory retinal degenerative disease, and induced by complex pathophysiological conditions. It is necessary to consider further the most efficacious combinations of optimal drugs, doses, routes, and drug release patterns (sustained-release, pulsatile-release, or controlled-release by responding to a trigger) based on the pathophysiology and progressive courses of the targeted disease.

#### **10. References**

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

membrane totally disappeared at 8 weeks. During 6 months of follow-up, no signs of

A microelectromechanical systems (MEMS) drug delivery device is investigated for the treatment of chronic and refractory ocular diseases (Lo et al., 2009, Saati et al., 2010). MEMS device can be re-filled with the drug solution, giving long-term drug therapy which avoids repeated surgeries. The first generation of MEMS is a manually-controlled system limited by variations in the drug-release duration and force applied for depressing of the reservoir. To resolve this problem, the next generation device consists of an electrolysis chamber with electrolysis actuation to precisely delivery the desired dosage volume, a drug reservoir with refill port, battery and electronics. Biocompatible and flexible parylene is used to construct the MEMS. Battery and wireless inductive power transfer can be used to drive electrolysis. Electrolysis is a low power process in which the electrochemically-induced phase change of water to hydrogen and oxygen gas generates pressure in the reservoir forcing the drug through the cannula (Saati et al., 2010). The reservoir is implanted in the subconjunctival space and flexible cannula is inserted through incision into the anterior or posterior segment. Gonzalez-Soto et al. have demonstrated that a slower prolonged infusion of the same volume and concentration of intravitreal ranibizumab is equivalent to a bolus intravitreal injection of ranibizumab to human VEGF-induced retinal hyperpermeability

Replenish, Inc. (Pasadena, CA, U.S.) plans to enter clinical trials for a refillable and programmable pump that would be implanted in the eye to feed medicine for glaucoma or AMD. The Replenish device can last more than 5 years before needing replacement, much

On Demand Therapeutics, Inc. (Menlo Park, CA, U.S.) has been developing a multi-reservoir implantable device for laser-activated drug delivery to the posterior segment of the eye (RetinaToday, 2010) (On Demand Therapeutics Inc). The injectable, biocompatible, nonresorbable device (ODTx) contains reservoirs designed for drug release in optimized doses. The reservoirs are capable of storing small- or large-molecule drugs that can be released via a standard, non-invasive laser activation procedure. The multiple reservoir system allows for ophthalmologists to control drug delivery by activating specific reservoirs, while unactivated reservoirs remain intact. Unfortunately, clinical trials of ODTx have not

Recent advances in drug delivery systems under clinical situation and in the late experimental stages are described in this chapter. AMD is chronic, progressive and refractory retinal degenerative disease, and induced by complex pathophysiological conditions. It is necessary to consider further the most efficacious combinations of optimal drugs, doses, routes, and drug release patterns (sustained-release, pulsatile-release, or

recurrence were observed.

**8.2 Micropump™ system** 

model of rabbits (Gonzalez-Soto et al., 2011).

longer than current treatments (Flanigan, 2009).

**8.3 ODTx** 

progressed yet.

**9. Conclusion** 


Clinical Application of Drug Delivery Systems for Treating AMD 243

ClinicalTrials.gov. NCT00791570, Anti-VEGFR vaccine therapy in treating patients with

<http://clinicaltrials.gov/ct2/show/NCT00791570?term=Osaka+University&rank

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Date, Y., Kimura, A., Kato, H. & Sasazuki, T. (1996). DNA typing of the HLA-A gene:

de Freitas, R. L., Santos, I. M., de Souza, G. F., Tome Ada, R., Saldanha, G. B. & de Freitas, R.

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**13**

**Use of OCT Imaging in the**

Simona-Delia Ţălu1 and Ştefan Ţălu2

*Faculty of Mechanics* 

*Romania* 

**Diagnosis and Monitoring of**

**Age Related Macular Degeneration** 

*1"Iuliu Haţieganu" University of Medicine and Pharmacy, Cluj-Napoca/Ophthalmology 2Technical University, Cluj-Napoca/Descriptive Geometry and Engineering Graphics,* 

Optical Coherence Tomography (OCT) is a non-invasive, high-resolution imaging technique that has been introduced in the clinical practice at the beginning of the last decade. The first application of this method has been recorded in the field of ophthalmology (Osiac et al., 2011). Retinal diseases such as Age-related Macular Degeneration (AMD), central serous chorioretinopathy, macular hole, vitreo-macular interface syndrome and diabetic maculopathy have taken advantage of this relatively new imaging method. Among these, AMD is by far, the ocular condition that has benefited the most from the enormous advantages offered by OCT, in terms of diagnosis, response to treatment and monitoring. Future progress in OCT techniques is expected to improve the knowledge in the pathophysiology of this devastating disease. In order to better understand the role of OCT in the management of AMD, a concise review of the physical principles and mathematical equations that sustain this method is provided. The progress in the OCT techniques over the past decade is emphasized, from Time Domain – OCT (TD-OCT) to Spectral Domain – OCT (SD-OCT) and future directions, with implications in the clinical practice. The comparative contribution of TD-OCT and SD-OCT in the different forms of AMD is revealed. The limits of OCT are presented with their possible solutions. After the description of the theoretical data for OCT interpretation, the impact of OCT in the diagnosis is illustrated with examples of various aspects that AMD can display. The role of OCT in the monitoring of AMD is revealed by the response of the wet form of the

The development of the retinal imaging was dued to three major events. The first one was represented by the invention of the direct ophthalmoscope by Hermann von Helmholtz in 1851: it opened the field of ocular imaging, by allowing the physicians to examine the retina in vivo. The second one took place in 1961, when Novotny and Davis used fluorescein dye to visualize the retinal circulation, inventing the technique named fluorescein angiography. The third major breakthrough happened in the 1990s, when OCT was invented, providing a

**1. Introduction** 

disease to the anti-VEGF intravitreal injections.

**2. OCT imaging in the diagnosis and monitoring of AMD** 


## **Use of OCT Imaging in the Diagnosis and Monitoring of Age Related Macular Degeneration**

Simona-Delia Ţălu1 and Ştefan Ţălu2

*1"Iuliu Haţieganu" University of Medicine and Pharmacy, Cluj-Napoca/Ophthalmology 2Technical University, Cluj-Napoca/Descriptive Geometry and Engineering Graphics, Faculty of Mechanics Romania* 

#### **1. Introduction**

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

Zhang, C., Shen, J. K., Lam, T. T., Zeng, H. Y., Chiang, S. K., Yang, F. & Tso, M. O. (2005).

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6245

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photoreceptors by BDNF gene transfer using in vivo electroporation in the RCS rat

Optical Coherence Tomography (OCT) is a non-invasive, high-resolution imaging technique that has been introduced in the clinical practice at the beginning of the last decade. The first application of this method has been recorded in the field of ophthalmology (Osiac et al., 2011). Retinal diseases such as Age-related Macular Degeneration (AMD), central serous chorioretinopathy, macular hole, vitreo-macular interface syndrome and diabetic maculopathy have taken advantage of this relatively new imaging method. Among these, AMD is by far, the ocular condition that has benefited the most from the enormous advantages offered by OCT, in terms of diagnosis, response to treatment and monitoring. Future progress in OCT techniques is expected to improve the knowledge in the pathophysiology of this devastating disease. In order to better understand the role of OCT in the management of AMD, a concise review of the physical principles and mathematical equations that sustain this method is provided. The progress in the OCT techniques over the past decade is emphasized, from Time Domain – OCT (TD-OCT) to Spectral Domain – OCT (SD-OCT) and future directions, with implications in the clinical practice. The comparative contribution of TD-OCT and SD-OCT in the different forms of AMD is revealed. The limits of OCT are presented with their possible solutions. After the description of the theoretical data for OCT interpretation, the impact of OCT in the diagnosis is illustrated with examples of various aspects that AMD can display. The role of OCT in the monitoring of AMD is revealed by the response of the wet form of the disease to the anti-VEGF intravitreal injections.

#### **2. OCT imaging in the diagnosis and monitoring of AMD**

The development of the retinal imaging was dued to three major events. The first one was represented by the invention of the direct ophthalmoscope by Hermann von Helmholtz in 1851: it opened the field of ocular imaging, by allowing the physicians to examine the retina in vivo. The second one took place in 1961, when Novotny and Davis used fluorescein dye to visualize the retinal circulation, inventing the technique named fluorescein angiography. The third major breakthrough happened in the 1990s, when OCT was invented, providing a

Use of OCT Imaging in the

in the sample (Talu et al., 2009).

evident with conventional 2D images (Talu et al., 2009).

**2.2.1 Time-domain OCT (TD-OCT) or conventional OCT** 

Fig. 1. A simplified schematic of Time domain (TD-OCT)

Diagnosis and Monitoring of Age Related Macular Degeneration 255

axial and lateral resolution, cross-sectional (2D) scan, 3D raster scanning and a higher imaging sensitivity than the traditional TD-OCT units. The SD-OCT software permits many operations with 3D data compared with traditional TD-OCT. The great number of scans done per time unit also allows SD-OCT systems to generate 3D reconstructions, which can be further manipulated. Visualization of this data in 3D demonstrates subtle pathology not

A superluminiscent diode emits a light beam which is split into two beams: a beam that enters the eye and is reflected back by the ocular media and a beam reflected by a reference mirror. The two beams meet, generating interferences that are intercepted by a light detector (fig. 1). By displacing the reference mirror, different structures, located at various depths, can be analyzed, thus obtaining an A-scan. The transverse scanning of the retina in a predefined axis (horizontal, vertical or oblique) is generating the B-scan of the retina, composed by the A-scan sequences. There are two important specifications concerning the quality of the image obtained: it depends on the number of retinal scans and partly, on the degree of light absorption by various retinal and subretinal structures. The time which is required in order to get the sections is the main determinant of the quality of the signal, justifying the name of Time Domain which is given to this OCT method. Time domain OCT (TD-OCT) is a technique that produces two-dimensional images of the sample internal structure. In standard TD-OCT two different scanning procedures are used in order to obtain an image: a depth scan using time-domain low coherence interferometry and a lateral scan addressing laterally adjacent positions to obtain the location of light scattering bodies

In TD-OCT, light from the low-coherence light source is divided evenly by the beam splitter. Half the light from the beam splitter is directed toward the sample and half the light toward a moving mirror. Light reflects off the mirror and from within the sample. The light beams reflected back are recombined by the beam splitter and directed into a detector. If the pathlengths match within a coherence length, interference will occur. OCT measures the intensity of interference and uses it to represent back-reflection intensity; the unwanted

fast, non-invasive, radiation-free examination technique, able to visualize precisely the retinal layers – something done before only on pathology slides. It's easy to understand why OCT has become soon an irreplaceable tool in most of the retina practices (Khaderi et al., 2011).

#### **2.1 Theoretical considerations on OCT**

Tomography is based on the reconstruction of cross-sectional images of an object using its projections. The concept of optical coherence tomography (OCT) was developed at Massachusetts Institute of Technology in the early 1990s and the first commercial version of OCT was made available by Carl Zeiss (Jena, Germany) in 1996. OCT is an extension of optical coherence domain reflectometry (OCDR). OCT is a modern noninvasive imaging technique with a high depth resolution, based on low coherence interferometry (LCI), that is able to reconstruct (tomographic) sectional images of the object under study. The first application of LCI in ophthalmology was to measure the eye length. OCT is similar to ultrasound imaging, but as an optical echo technique has much higher resolution. The key benefits of OCT are: live sub-surface images at near-microscopic resolution; instant, direct imaging of tissue morphology; no preparation of the sample or subject; no ionizing radiation. OCT is useful in situations where biopsy can't be performed, where sampling areas with conventional biopsies are likely, and that involve guiding surgical / microsurgical procedures.

The most important advantage of OCT as a diagnostic tool in ophthalmology is the obtain of fast, non-contact images of the ocular structures such as cornea, lens, retina and the optic nerve with depth resolutions better than 3 μm. Thus it is used to obtain a cross section of the retina based on the reflectivity on different layers within the retina, allowing detection of morphologic and micrometric modifications in retinal tissue. The ability to measure thickness of retinal layers has potential for early detection of pathologies and disease diagnosis (Talu et al., 2009).

#### **2.2 OCT methods**

OCT is applied by two main methods: Time domain OCT (TD-OCT) and Spectral domain OCT (SD-OCT). Each method has its own advantages and limitations.

TD-OCT produces two-dimensional images of the sample internal structure. In TD-OCT, tissue-reflectance information in depth (an A-scan represents a reflectivity profile in depth) is gradually built up over time by moving a mirror in the reference arm of the interferometer. OCT B-scans (a B-scan represents a cross-section image, a lateral x depth map) are generated by collecting many A-scans (Walther et al., 2011).

SD-OCT can be implemented in two formats, Fourier domain (FD-OCT) and swept source (SS-OCT). SD-OCT units acquire entire A-scans in reflected light at a given point in tissue. Information on depth is transformed from the frequency domain to the time domain, without using a moving reference mirror to obtain complete A-scans. The absence of moving parts allows the image to be acquired rapidly - about 60 times faster than with TD-OCT (Walther et al., 2011). The SD-OCT units allow the improvement of the detection and monitoring of retinal diseases, because these ones have ultra high-speed scan rate, superior

fast, non-invasive, radiation-free examination technique, able to visualize precisely the retinal layers – something done before only on pathology slides. It's easy to understand why OCT has become soon an irreplaceable tool in most of the retina practices (Khaderi et al.,

Tomography is based on the reconstruction of cross-sectional images of an object using its projections. The concept of optical coherence tomography (OCT) was developed at Massachusetts Institute of Technology in the early 1990s and the first commercial version of OCT was made available by Carl Zeiss (Jena, Germany) in 1996. OCT is an extension of optical coherence domain reflectometry (OCDR). OCT is a modern noninvasive imaging technique with a high depth resolution, based on low coherence interferometry (LCI), that is able to reconstruct (tomographic) sectional images of the object under study. The first application of LCI in ophthalmology was to measure the eye length. OCT is similar to ultrasound imaging, but as an optical echo technique has much higher resolution. The key benefits of OCT are: live sub-surface images at near-microscopic resolution; instant, direct imaging of tissue morphology; no preparation of the sample or subject; no ionizing radiation. OCT is useful in situations where biopsy can't be performed, where sampling areas with conventional biopsies are likely, and that involve guiding surgical /

The most important advantage of OCT as a diagnostic tool in ophthalmology is the obtain of fast, non-contact images of the ocular structures such as cornea, lens, retina and the optic nerve with depth resolutions better than 3 μm. Thus it is used to obtain a cross section of the retina based on the reflectivity on different layers within the retina, allowing detection of morphologic and micrometric modifications in retinal tissue. The ability to measure thickness of retinal layers has potential for early detection of pathologies and disease

OCT is applied by two main methods: Time domain OCT (TD-OCT) and Spectral domain

TD-OCT produces two-dimensional images of the sample internal structure. In TD-OCT, tissue-reflectance information in depth (an A-scan represents a reflectivity profile in depth) is gradually built up over time by moving a mirror in the reference arm of the interferometer. OCT B-scans (a B-scan represents a cross-section image, a lateral x depth

SD-OCT can be implemented in two formats, Fourier domain (FD-OCT) and swept source (SS-OCT). SD-OCT units acquire entire A-scans in reflected light at a given point in tissue. Information on depth is transformed from the frequency domain to the time domain, without using a moving reference mirror to obtain complete A-scans. The absence of moving parts allows the image to be acquired rapidly - about 60 times faster than with TD-OCT (Walther et al., 2011). The SD-OCT units allow the improvement of the detection and monitoring of retinal diseases, because these ones have ultra high-speed scan rate, superior

OCT (SD-OCT). Each method has its own advantages and limitations.

map) are generated by collecting many A-scans (Walther et al., 2011).

2011).

**2.1 Theoretical considerations on OCT** 

microsurgical procedures.

diagnosis (Talu et al., 2009).

**2.2 OCT methods** 

axial and lateral resolution, cross-sectional (2D) scan, 3D raster scanning and a higher imaging sensitivity than the traditional TD-OCT units. The SD-OCT software permits many operations with 3D data compared with traditional TD-OCT. The great number of scans done per time unit also allows SD-OCT systems to generate 3D reconstructions, which can be further manipulated. Visualization of this data in 3D demonstrates subtle pathology not evident with conventional 2D images (Talu et al., 2009).

#### **2.2.1 Time-domain OCT (TD-OCT) or conventional OCT**

A superluminiscent diode emits a light beam which is split into two beams: a beam that enters the eye and is reflected back by the ocular media and a beam reflected by a reference mirror. The two beams meet, generating interferences that are intercepted by a light detector (fig. 1). By displacing the reference mirror, different structures, located at various depths, can be analyzed, thus obtaining an A-scan. The transverse scanning of the retina in a predefined axis (horizontal, vertical or oblique) is generating the B-scan of the retina, composed by the A-scan sequences. There are two important specifications concerning the quality of the image obtained: it depends on the number of retinal scans and partly, on the degree of light absorption by various retinal and subretinal structures. The time which is required in order to get the sections is the main determinant of the quality of the signal, justifying the name of Time Domain which is given to this OCT method. Time domain OCT (TD-OCT) is a technique that produces two-dimensional images of the sample internal structure. In standard TD-OCT two different scanning procedures are used in order to obtain an image: a depth scan using time-domain low coherence interferometry and a lateral scan addressing laterally adjacent positions to obtain the location of light scattering bodies in the sample (Talu et al., 2009).

Fig. 1. A simplified schematic of Time domain (TD-OCT)

In TD-OCT, light from the low-coherence light source is divided evenly by the beam splitter. Half the light from the beam splitter is directed toward the sample and half the light toward a moving mirror. Light reflects off the mirror and from within the sample. The light beams reflected back are recombined by the beam splitter and directed into a detector. If the pathlengths match within a coherence length, interference will occur. OCT measures the intensity of interference and uses it to represent back-reflection intensity; the unwanted

Use of OCT Imaging in the

Diagnosis and Monitoring of Age Related Macular Degeneration 257

the light source. As explained above, the SD-OCT devices use a broadband light source and a spectrometer, generating readout rates of 40 kHz. FD-OCT utilizes a wavelength-swept laser source, which allows the obtaining of repetition rates as high as 370 kHz. The clinical potential of FD-OCTs is vast, given to their wide fields of view: for instance, they reach the

**Feature TD-OCT SD-OCT** 

The Fourier transformation allows the **simultaneous** measurement of the light reflection with a spectrometer

It samples all the points simultaneously 60 times faster than TD-OCT

65.000 scans in a 6 mm area, without excluding areas

scans/second

3D reconstruction possible

An interferometer measures **sequentially** the echo delay time of light that is reflected by the retinal microstructures.

It samples one point at the time

6 radial scans, 20 μ wide and 6 mm long (the area between the 6 scans is not imaged)

**Rate of acquisition** 400 scans/second 20.000 – 40.000

Two-dimensional images of the sample internal structure

**and quality** 10 μ 1 μ, clearer

The antero-posterior sections on OCT reveal the succession of the retinal layers and of the Retinal Pigmented Epithelium (RPE), as well as the presence of any spaces between these

Retinal thickening (the thickness of the retina is measured between the internal limiting membrane and the RPE) is determined by the exudation from the choroidal new vessels.

The occult Choroidal Neovascularization (CNV) is revealed by the constant presence of the elevation or detachment of the RPE band. Frequently, the occult CNV is suggested by

various alteration of the RPE: irregularities, fragmentation, thickening, thinning.

layers. The information offered by OCT is detailed, simple and easily interpretable.

The OCT signs in AMD are extremely valuable for the ophthalmologist.

coronary arteries and esophageal epithelium (Khaderi et al., 2011).

**Basis** 

**Modality of sampling** 

**Scanned area** 

**Result** 

**Image resolution** 

Table 1. Comparison of TD-OCT vs. SD-OCT

**2.3 Clinical applications of OCT in AMD** 

**2.3.1 Basis of OCT interpretation** 

background light is suppressed by filtering. The OCT signal contains the oscillating term of the intensity, that is the integral of the contribution of the light reflected back from the biological tissue at all depths. TD-OCT uses light to map the layers within the retina in a cross-sectional image. Each image is made up of a series of A-scans scanned through the depth of the tissue and when aligned side by side, creates a B-scan two dimensional crosssectional image. Each A-scan is acquired by moving a reference mirror to correspond to each point along the depth of the A-scan, and the signals from the reference arm and from the retina are interfered to determine the signal at that point in the A-scan. A TD-OCT system has two major spatial resolutions: axial and transversal resolution. The axial resolution depends on two laser characteristics: coherence length of the light source and pulse duration. The transversal resolution is determined by the focused spot size of the optical beam. The capability of TD-OCT system to produce images with good quality is characterized by the value of the signal to noise ratio (SNR) (Talu et al., 2009).

#### **2.2.2 Spectral-domain OCT (SD-OCT)**

The development of the SD-OCT is originating in the Fourier mathematical equation (1807) that can sum a periodic function into a series of sinusoidal functions. When applied to the OCT, the Fourier transformation replaces the sequential measurement of the reflected beam (by moving a mirror in front of the reference beam), with the simultaneous measurement of the light reflection. Subsequently, the image resolution in SD-OCT is 1 μm, as compared to 10 μm in TD-OCT. The practical impact of this improvement in resolution is the early detection of small cystic changes associated with the wet form of AMD. The early diagnosis is followed by the early treatment and the better preservation of the visual function. In SD-OCT, a spectrometer is used in order to analyze simultaneously all the frequencies. Therefore, all echoes of light from the various layers of the retina can be measured simultaneously, making the image acquisition much more rapid: SD-OCT is 50 times faster than the conventional TD-OCT and 100 times faster than the first ultrahigh-resolution OCT (UHR-OCT). The axial depth depends mainly on the bandwidth of the light source. With UHR-OCT, the resolution is 2 – 3 μ (the standard is of 10 μ). This ultrahigh resolution is comparable with the one obtained in histopathology, which makes possible the earlier diagnosis in AMD, better guidance of treatment and improvement of knowledge in the pathogenesis of this disease (Coscas et al., 2009).

Given the possibility to simultaneously get images in various planes, the 3D reconstruction is possible with SD-OCT, allowing the obtaining of hundreds of high-resolution images per second. The reduction of the examination time considerably decreases the artifacts related to patient movements. The SD-OCT images have proven to be clearer and with higher quality as compared to the ones obtained by the successive TD-OCT systems (OCT1, OCT3, stratus). The SD-OCT systems are continuously improving, by adding complementary functions: fundus photography, angiography, microperimetry. The 3D evaluation permits the accurate measurement of the macula (total volume) in various conditions (edema, fluid, drusen, CNV) with implications in the follow up and treatment of AMD (Luviano et al., 2009). The ultra-high resolution images obtained by SD-OCT allow a better differentiation between the retinal and subretinal layers. Further research has led to another significant improvement in resolution and speed: the novel frequency-domain OCT devices (FD-OCT), which are not yet available for widespread use. The difference between SD-OCT and FD-OCT resides in

background light is suppressed by filtering. The OCT signal contains the oscillating term of the intensity, that is the integral of the contribution of the light reflected back from the biological tissue at all depths. TD-OCT uses light to map the layers within the retina in a cross-sectional image. Each image is made up of a series of A-scans scanned through the depth of the tissue and when aligned side by side, creates a B-scan two dimensional crosssectional image. Each A-scan is acquired by moving a reference mirror to correspond to each point along the depth of the A-scan, and the signals from the reference arm and from the retina are interfered to determine the signal at that point in the A-scan. A TD-OCT system has two major spatial resolutions: axial and transversal resolution. The axial resolution depends on two laser characteristics: coherence length of the light source and pulse duration. The transversal resolution is determined by the focused spot size of the optical beam. The capability of TD-OCT system to produce images with good quality is

The development of the SD-OCT is originating in the Fourier mathematical equation (1807) that can sum a periodic function into a series of sinusoidal functions. When applied to the OCT, the Fourier transformation replaces the sequential measurement of the reflected beam (by moving a mirror in front of the reference beam), with the simultaneous measurement of the light reflection. Subsequently, the image resolution in SD-OCT is 1 μm, as compared to 10 μm in TD-OCT. The practical impact of this improvement in resolution is the early detection of small cystic changes associated with the wet form of AMD. The early diagnosis is followed by the early treatment and the better preservation of the visual function. In SD-OCT, a spectrometer is used in order to analyze simultaneously all the frequencies. Therefore, all echoes of light from the various layers of the retina can be measured simultaneously, making the image acquisition much more rapid: SD-OCT is 50 times faster than the conventional TD-OCT and 100 times faster than the first ultrahigh-resolution OCT (UHR-OCT). The axial depth depends mainly on the bandwidth of the light source. With UHR-OCT, the resolution is 2 – 3 μ (the standard is of 10 μ). This ultrahigh resolution is comparable with the one obtained in histopathology, which makes possible the earlier diagnosis in AMD, better guidance of treatment and improvement of knowledge in the

Given the possibility to simultaneously get images in various planes, the 3D reconstruction is possible with SD-OCT, allowing the obtaining of hundreds of high-resolution images per second. The reduction of the examination time considerably decreases the artifacts related to patient movements. The SD-OCT images have proven to be clearer and with higher quality as compared to the ones obtained by the successive TD-OCT systems (OCT1, OCT3, stratus). The SD-OCT systems are continuously improving, by adding complementary functions: fundus photography, angiography, microperimetry. The 3D evaluation permits the accurate measurement of the macula (total volume) in various conditions (edema, fluid, drusen, CNV) with implications in the follow up and treatment of AMD (Luviano et al., 2009). The ultra-high resolution images obtained by SD-OCT allow a better differentiation between the retinal and subretinal layers. Further research has led to another significant improvement in resolution and speed: the novel frequency-domain OCT devices (FD-OCT), which are not yet available for widespread use. The difference between SD-OCT and FD-OCT resides in

characterized by the value of the signal to noise ratio (SNR) (Talu et al., 2009).

**2.2.2 Spectral-domain OCT (SD-OCT)** 

pathogenesis of this disease (Coscas et al., 2009).

the light source. As explained above, the SD-OCT devices use a broadband light source and a spectrometer, generating readout rates of 40 kHz. FD-OCT utilizes a wavelength-swept laser source, which allows the obtaining of repetition rates as high as 370 kHz. The clinical potential of FD-OCTs is vast, given to their wide fields of view: for instance, they reach the coronary arteries and esophageal epithelium (Khaderi et al., 2011).


Table 1. Comparison of TD-OCT vs. SD-OCT

#### **2.3 Clinical applications of OCT in AMD**

The antero-posterior sections on OCT reveal the succession of the retinal layers and of the Retinal Pigmented Epithelium (RPE), as well as the presence of any spaces between these layers. The information offered by OCT is detailed, simple and easily interpretable.

#### **2.3.1 Basis of OCT interpretation**

The OCT signs in AMD are extremely valuable for the ophthalmologist.

Retinal thickening (the thickness of the retina is measured between the internal limiting membrane and the RPE) is determined by the exudation from the choroidal new vessels.

The occult Choroidal Neovascularization (CNV) is revealed by the constant presence of the elevation or detachment of the RPE band. Frequently, the occult CNV is suggested by various alteration of the RPE: irregularities, fragmentation, thickening, thinning.

Use of OCT Imaging in the

thickness (Coscas et al., 2009).

OCT and SD-SLO/OCT (Forte et al., 2009).

**2.3.4 The segmentation of the retinal layers** 

**2.3.5 Drusen** 

Diagnosis and Monitoring of Age Related Macular Degeneration 259

high resolution scans obtained in SD-OCT allows the separation of the outer photoreceptor segments from the RPE and subsequently, their inclusion in the calculation of the retinal

In another recent study, it has been shown an increased measurement in retinal thickness of 65 – 70 μm as measured by Spectralis OCT compared with Stratus OCT which corresponds to the inclusion of the outer segment-RPE-Bruch's membrane complex by Spectralis OCT (Grover et al., 2010). Other studies proved the superiority of SD-OCT versus TD-OCT in quantifying the retinal thickness and evaluating the activity of the CNV membranes in wet AMD (Sayanagi et al, 2009; Mylonas et al, 2009). The differences between the macular thickness and volume measured with TD-OCT and SD-SLO (Scanning Laser Ophthalmoscopy)/OCT have been evaluated in eyes with macular edema and in normal eyes. The SD-SLO/OCT produced fewer artefacts than Stratus TD-OCT in normal and oedematous retina. Retinal thickness measured with SD-SLO/OCT has been significantly higher than retinal thickness obtained with TD-OCT. Therefore, it is advisable to follow the patient with the same OCT device, otherwise a correcting value of 1.1 should be considered when extrapolating the values from TD-OCT to SD-OCT. Retinal volume measurements were strongly reproducible and could be used to compare examinations with Stratus TD-

The interchangeability of retinal thickness measurements resulting from different protocols of Spectralis OCT (which combines the OCT with the confocal laser ophthalmoscopy) has been evaluated in healthy eyes. It showed good protocol interchangeability for all tested protocols, which is important as it allows the selection of a more rapid and simpler protocol, especially in less cooperative patients. Higher number of measurements might influence negatively the results due to corneal dryness and loss of attentiveness (Wenner et al., 2011).

TD-OCT can measure the thickness of the entire retina (from the vitreo-retinal interface to the RPE), but the only structure that can be isolated and measured individually is the nerve fiber layer. The TD-OCT conventional software does not allow the measurement of the structures that are external to the RPE: Pigment Epithelial Detachments (PED), CNV. Because the evolution in time of the subretinal space is extremely important for the monitoring and

SD-OCT, by facilitating the image segmentation, makes it possible to individualize certain layers of the retina: plexiform layer, subretinal space, subretinal pigment epithelial space. The segmentation of the retina into two components (neurosensory and subretinal space) is

The evaluation of drusen is important for the prediction of AMD risk for progression, despite the differences in agreement between observers. Whereas with TD-OCT the imaging of drusen is limited because of artifacts, the SD-OCT is capable to provide its detailed structure, thanks to the segmentation techniques. The internal structure of the drusen, that can now be specified, seems to be an indirect indicator of the complement-related activity which is associated with the risk of progression. However, this observation needs to be

decision making in AMD, they are measured with custom programs or manually.

also a promising option for the follow up of AMD (Coscas et al., 2009).

The subretinal fluid appears as diffuse infiltration or as the constitution of cystic spaces in the macular area.

The classic CNV is translated on OCT as hyper-reflective zones adjacent to or away from the RPE. They must be differentiated from other hyper-reflective structures: fibrous tissue, exudate, pigment, pseudo-vitelliform material.

Other signs of prognostic value can be visualized at the level of the outer retinal layers (outer nuclear layer and external limiting membrane): hyper-reflective spots and areas of densification. They prove the progression of the disease (Coscas et al., 2009).

#### **2.3.2 Technological parameters**

Axial resolution (depth) refers to the capability to measure the morphological architecture of each retinal layer.

The depth of penetration depends mainly on two parameters: the optical properties of the tissues and the imaging wavelength that is used. Studies are conducted in order to find a possibility to visualize the choroid.

Transverse resolution of the image could reach a level allowing to distinguish cells.

The sensitivity of detection is a measure of the ease to obtain good quality OCT images in case of ocular media opacities.

The data acquisition time settles the number of transverse pixels of the OCT image.

Image contrast is an additional parameter meant to improve the visualization of various structures.

Functional extensions of OCT have the purpose to offer an optical biopsy of the retina, simultaneously with functional and metabolic data on its activity (Coscas et al., 2009).

#### **2.3.3 Macula**

The most frequent application of OCT in retinal disease is the measurement and monitoring of the retinal thickness.

The TD - OCT system (Stratus) gives a macular thickness map which is calculated from 6 radial B-scans crossing at the fovea. By interpolating data from these scans, the average macular thickness is calculated in 9 subfields centered on the fovea. Similarly, the total macular volume is obtained.

The SD-OCT provides images with much higher resolution. Given the differences between the measurements with the two types of OCT (TD and SD), algorithms are necessary in order to establish correlations between them.

Various studies compared the retinal thickness measured with TD-OCT and SD-OCT. For instance, the comparative measurements of the retinal thickness performed with Cirrus HD-OCT and Stratus TD-OCT in healthy individuals revealed that the average retinal thickness measured with SD-OCT has been significantly higher as with TD-OCT: 60 μm thicker (Kakinoki et al., 2009). This is explained by the difference in defining the retinal thickness between the two machines. In TD-OCT, the outer segments of the photoreceptors are not differentiated from the RPE, thus being excluded from the retinal thickness evaluation. The

The subretinal fluid appears as diffuse infiltration or as the constitution of cystic spaces in

The classic CNV is translated on OCT as hyper-reflective zones adjacent to or away from the RPE. They must be differentiated from other hyper-reflective structures: fibrous tissue,

Other signs of prognostic value can be visualized at the level of the outer retinal layers (outer nuclear layer and external limiting membrane): hyper-reflective spots and areas of

Axial resolution (depth) refers to the capability to measure the morphological architecture of

The depth of penetration depends mainly on two parameters: the optical properties of the tissues and the imaging wavelength that is used. Studies are conducted in order to find a

The sensitivity of detection is a measure of the ease to obtain good quality OCT images in

Image contrast is an additional parameter meant to improve the visualization of various

Functional extensions of OCT have the purpose to offer an optical biopsy of the retina, simultaneously with functional and metabolic data on its activity (Coscas et al., 2009).

The most frequent application of OCT in retinal disease is the measurement and monitoring

The TD - OCT system (Stratus) gives a macular thickness map which is calculated from 6 radial B-scans crossing at the fovea. By interpolating data from these scans, the average macular thickness is calculated in 9 subfields centered on the fovea. Similarly, the total

The SD-OCT provides images with much higher resolution. Given the differences between the measurements with the two types of OCT (TD and SD), algorithms are necessary in

Various studies compared the retinal thickness measured with TD-OCT and SD-OCT. For instance, the comparative measurements of the retinal thickness performed with Cirrus HD-OCT and Stratus TD-OCT in healthy individuals revealed that the average retinal thickness measured with SD-OCT has been significantly higher as with TD-OCT: 60 μm thicker (Kakinoki et al., 2009). This is explained by the difference in defining the retinal thickness between the two machines. In TD-OCT, the outer segments of the photoreceptors are not differentiated from the RPE, thus being excluded from the retinal thickness evaluation. The

Transverse resolution of the image could reach a level allowing to distinguish cells.

The data acquisition time settles the number of transverse pixels of the OCT image.

densification. They prove the progression of the disease (Coscas et al., 2009).

the macular area.

each retinal layer.

structures.

**2.3.3 Macula** 

of the retinal thickness.

macular volume is obtained.

order to establish correlations between them.

exudate, pigment, pseudo-vitelliform material.

**2.3.2 Technological parameters** 

possibility to visualize the choroid.

case of ocular media opacities.

high resolution scans obtained in SD-OCT allows the separation of the outer photoreceptor segments from the RPE and subsequently, their inclusion in the calculation of the retinal thickness (Coscas et al., 2009).

In another recent study, it has been shown an increased measurement in retinal thickness of 65 – 70 μm as measured by Spectralis OCT compared with Stratus OCT which corresponds to the inclusion of the outer segment-RPE-Bruch's membrane complex by Spectralis OCT (Grover et al., 2010). Other studies proved the superiority of SD-OCT versus TD-OCT in quantifying the retinal thickness and evaluating the activity of the CNV membranes in wet AMD (Sayanagi et al, 2009; Mylonas et al, 2009). The differences between the macular thickness and volume measured with TD-OCT and SD-SLO (Scanning Laser Ophthalmoscopy)/OCT have been evaluated in eyes with macular edema and in normal eyes. The SD-SLO/OCT produced fewer artefacts than Stratus TD-OCT in normal and oedematous retina. Retinal thickness measured with SD-SLO/OCT has been significantly higher than retinal thickness obtained with TD-OCT. Therefore, it is advisable to follow the patient with the same OCT device, otherwise a correcting value of 1.1 should be considered when extrapolating the values from TD-OCT to SD-OCT. Retinal volume measurements were strongly reproducible and could be used to compare examinations with Stratus TD-OCT and SD-SLO/OCT (Forte et al., 2009).

The interchangeability of retinal thickness measurements resulting from different protocols of Spectralis OCT (which combines the OCT with the confocal laser ophthalmoscopy) has been evaluated in healthy eyes. It showed good protocol interchangeability for all tested protocols, which is important as it allows the selection of a more rapid and simpler protocol, especially in less cooperative patients. Higher number of measurements might influence negatively the results due to corneal dryness and loss of attentiveness (Wenner et al., 2011).

#### **2.3.4 The segmentation of the retinal layers**

TD-OCT can measure the thickness of the entire retina (from the vitreo-retinal interface to the RPE), but the only structure that can be isolated and measured individually is the nerve fiber layer. The TD-OCT conventional software does not allow the measurement of the structures that are external to the RPE: Pigment Epithelial Detachments (PED), CNV. Because the evolution in time of the subretinal space is extremely important for the monitoring and decision making in AMD, they are measured with custom programs or manually.

SD-OCT, by facilitating the image segmentation, makes it possible to individualize certain layers of the retina: plexiform layer, subretinal space, subretinal pigment epithelial space. The segmentation of the retina into two components (neurosensory and subretinal space) is also a promising option for the follow up of AMD (Coscas et al., 2009).

#### **2.3.5 Drusen**

The evaluation of drusen is important for the prediction of AMD risk for progression, despite the differences in agreement between observers. Whereas with TD-OCT the imaging of drusen is limited because of artifacts, the SD-OCT is capable to provide its detailed structure, thanks to the segmentation techniques. The internal structure of the drusen, that can now be specified, seems to be an indirect indicator of the complement-related activity which is associated with the risk of progression. However, this observation needs to be

Use of OCT Imaging in the

AMD modifications.

Diagnosis and Monitoring of Age Related Macular Degeneration 261

angiography, SLO, Eye -Tracking systems, microperimetry (Marschall et al., 2011). The use of various wavelengths would create the possibility to penetrate deeper and examine the choroid. The improvement of the possibilities to process the 3D images will allow the more

**2.4 Personal experience with OCT imaging in the diagnosis and monitoring of AMD**  The contribution of OCT in the daily practice is illustrated by several examples of various

FD-OCT imaging was performed with a Topcon 3D OCT-1000 instrument (TOPCON, Japan, model 2007) that uses a monochromatic light source of 840 nm wave length. The Topcon 3D

In fig. 2a appears the image of a normal fundus, showing the optic nerve head, the macular region and the retinal vessels (arteries and veins). Fig. 2b displays the 2D FD-OCT image of the macula, showing (from the surface to the deep layers): the foveolar depression, the neurosensory retina and the RPE (the red layer). Fig. 2c illustrates the 3D FD-OCT image of the macula, revealing the architecture of the normal macular region, tridimensionally - a true "live biopsy" of the tissues. The tissues with high reflectivity appear more red on the OCT. For instance, on the normal OCT, the RPE band has the highest reflectivity (red color).

Fig. 2. The normal macula: a) ocular fundus; b) 2D scan; c) 3D scan (courtesy of Dr. F. Balta,

Eye Hospital, Bucharest, Romania)

OCT-1000 combines FD-OCT system with a color non-mydriatic retinal camera.

detailed and precise evaluation of a certain structure (Coscas et al., 2009).

**2.4.1 Fourier Domain-OCT (FD-OCT) in the diagnosis of AMD** 

validated by clinical studies. The possibility to precisely measure the drusen volume with the support of the computer-assisted techniques offers a very useful tool to monitor the disease and to assess the risk of AMD progression (Coscas et al., 2009).

#### **2.3.6 Choroidal neovascularization**

Conventional TD-OCT represents a reference moment in the retinal imaging by having made it possible to: quantify the retinal response to CNV (macular edema, subretinal fluid); establish a correlation between the morphological (cystoid macular edema) and functional (visual acuity) parameters and between the OCT measurements and the response to treatment; visualize the fibro-vascular membranes in the subretinal and sub-RPE space.

The advantages of SD-OCT are represented by: the possibility to generate 3D images of the fibro-vascular complexes and to correlate them with fluorescein angiography and microperimetry; the easier detection of small PED, CNV and subretinal fluid. These advantages are particularly important for the identification of the chorio-retinal anastomoses and for the management of higher precision clinical studies (Coscas et al., 2009).

#### **2.3.7 Geographic atrophy**

Areas of geographic atrophy are evaluated in clinical studies by color photographs and autofluorescence. In TD-OCT, the RPE atrophy can be revealed by the increased choroidal hyper-reflectivity. In SD-OCT, the improved transverse resolution differentiates more clearly the limits between the normal and abnormal RPE and the segmentation techniques allow the correlations of the RPE changes with the photoreceptor layer (Coscas et al., 2009).

#### **2.3.8 Therapeutical impact of OCT**

Besides its contribution in the diagnosis of AMD, OCT examination is extremely useful in establishing the indication of the modern treatments in wet AMD (intravitreal injections with anti-VEGF agents) and in the monitoring of the response to treatment. The clinical experience has set up an algorithm of follow-up and treatment, although the number of injections is not yet established. However, it seems that the recurrence rate decreases with time (Coscas et al., 2009).

#### **2.3.9 Limits of OCT**

OCT cannot precisely describe a CNV network, nor can it define its nature: active or prefibrotic. Therefore, the OCT scan must be interpreted in correlation with the fundus photography, direction of scan and ideally, the angiography (Coscas et al., 2009).

Another limitation of OCT is revealed in evaluating the extension of the geographic atrophy (GA). In a recent study, the GA areas identified in SLO scans were significantly larger than the ones detected on the OCT maps. Spectralis OCT showed significantly more mild and severe segmentation errors than 3D and Cirrus OCT. Taking into account the fact that GA is a frequent form of AMD, this limitation should be resolved in order to identify and document RPE loss in a realistic manner (Schutze et al., 2011).

#### **2.3.10 Future developments of OCT**

One of the most recent innovations is the possibility to deliver simultaneously data offered by various examination methods: OCT, red-free photography, autofluorescence,

validated by clinical studies. The possibility to precisely measure the drusen volume with the support of the computer-assisted techniques offers a very useful tool to monitor the

Conventional TD-OCT represents a reference moment in the retinal imaging by having made it possible to: quantify the retinal response to CNV (macular edema, subretinal fluid); establish a correlation between the morphological (cystoid macular edema) and functional (visual acuity) parameters and between the OCT measurements and the response to treatment; visualize the fibro-vascular membranes in the subretinal and sub-RPE space.

The advantages of SD-OCT are represented by: the possibility to generate 3D images of the fibro-vascular complexes and to correlate them with fluorescein angiography and microperimetry; the easier detection of small PED, CNV and subretinal fluid. These advantages are particularly important for the identification of the chorio-retinal anastomoses

Areas of geographic atrophy are evaluated in clinical studies by color photographs and autofluorescence. In TD-OCT, the RPE atrophy can be revealed by the increased choroidal hyper-reflectivity. In SD-OCT, the improved transverse resolution differentiates more clearly the limits between the normal and abnormal RPE and the segmentation techniques allow the correlations of the RPE changes with the photoreceptor layer (Coscas et al., 2009).

Besides its contribution in the diagnosis of AMD, OCT examination is extremely useful in establishing the indication of the modern treatments in wet AMD (intravitreal injections with anti-VEGF agents) and in the monitoring of the response to treatment. The clinical experience has set up an algorithm of follow-up and treatment, although the number of injections is not yet established. However, it seems that the recurrence rate decreases with time (Coscas et al., 2009).

OCT cannot precisely describe a CNV network, nor can it define its nature: active or prefibrotic. Therefore, the OCT scan must be interpreted in correlation with the fundus

Another limitation of OCT is revealed in evaluating the extension of the geographic atrophy (GA). In a recent study, the GA areas identified in SLO scans were significantly larger than the ones detected on the OCT maps. Spectralis OCT showed significantly more mild and severe segmentation errors than 3D and Cirrus OCT. Taking into account the fact that GA is a frequent form of AMD, this limitation should be resolved in order to identify and

One of the most recent innovations is the possibility to deliver simultaneously data offered by various examination methods: OCT, red-free photography, autofluorescence,

photography, direction of scan and ideally, the angiography (Coscas et al., 2009).

document RPE loss in a realistic manner (Schutze et al., 2011).

and for the management of higher precision clinical studies (Coscas et al., 2009).

disease and to assess the risk of AMD progression (Coscas et al., 2009).

**2.3.6 Choroidal neovascularization** 

**2.3.7 Geographic atrophy** 

**2.3.9 Limits of OCT** 

**2.3.8 Therapeutical impact of OCT** 

**2.3.10 Future developments of OCT** 

angiography, SLO, Eye -Tracking systems, microperimetry (Marschall et al., 2011). The use of various wavelengths would create the possibility to penetrate deeper and examine the choroid. The improvement of the possibilities to process the 3D images will allow the more detailed and precise evaluation of a certain structure (Coscas et al., 2009).

#### **2.4 Personal experience with OCT imaging in the diagnosis and monitoring of AMD**

The contribution of OCT in the daily practice is illustrated by several examples of various AMD modifications.

#### **2.4.1 Fourier Domain-OCT (FD-OCT) in the diagnosis of AMD**

FD-OCT imaging was performed with a Topcon 3D OCT-1000 instrument (TOPCON, Japan, model 2007) that uses a monochromatic light source of 840 nm wave length. The Topcon 3D OCT-1000 combines FD-OCT system with a color non-mydriatic retinal camera.

In fig. 2a appears the image of a normal fundus, showing the optic nerve head, the macular region and the retinal vessels (arteries and veins). Fig. 2b displays the 2D FD-OCT image of the macula, showing (from the surface to the deep layers): the foveolar depression, the neurosensory retina and the RPE (the red layer). Fig. 2c illustrates the 3D FD-OCT image of the macula, revealing the architecture of the normal macular region, tridimensionally - a true "live biopsy" of the tissues. The tissues with high reflectivity appear more red on the OCT. For instance, on the normal OCT, the RPE band has the highest reflectivity (red color).

Fig. 2. The normal macula: a) ocular fundus; b) 2D scan; c) 3D scan (courtesy of Dr. F. Balta, Eye Hospital, Bucharest, Romania)

Use of OCT Imaging in the

Fig. 4. HD-5 line raster: Drusen and PEDs (courtesy of Dr. H. Shah, Midland, Texas, USA)

diagnosis of the wet AMD.

8, and 9.

Diagnosis and Monitoring of Age Related Macular Degeneration 263

RPE detachment is characterized by the accumulation of fluid between the highly reflective RPE (red line) and the moderately reflective choriocapillaris. Blister-like elevation of the retina and RPE are evident on the OCT image. Figures 4 and 5 depict the PEDs: the red line representing the RPE is irregular and present focal elevations. Fig. 4 also shows the fluid

Occult CNV is a term given to a specific "blothcy" appearance on the angiogram. The occult CNV in fig. 6 is suggested by the irregularities and thickening of the RPE layer and the CME appears like small, cystic spaces within the macula. In the figure above, the occult CNV is not accompanied by PED, therefore it is cathegorized as type 2 occult CNV. The type 1 occult CNV associates PEDs and is also named vascularized PED. The occult CNV might be an early phase of the classic CNV and therefore its identification is important for the early

When the choroidal new vessels that have grown under the macula are seen on the angiogram, this condition is named classic CNV. The classic CNV appears on OCT as a hyper-reflective structure that elevates the RPE. This aspect is easily demonstrated in fig. 7,

accumulation in the retina in a region adjacent to the RPE detachment.

In fig. 3a, the ophthalmoscopic aspect of a wet form of AMD is illustrated: subfoveal neovascular membrane, elevating the retina, on average 1,5 disc diameters in surface. Fig. 3b depicts the 2D FD-OCT image of the macula in wet AMD: elevation of the macular retina, the space under the neurosensory retina is occupied by an irregular, opaque structure; the retinal pigmented epithelium is thickened. The 3D FD-OCT image of the macula in wet AMD (fig. 3c) offers a tridimensional view: elevation of the macular retina, better depiction of the retinal topography, thus improving the visualization of RPE irregularities. The advantage of FD-OCT in AMD is revealed by the fact that because it more densely samples the macula, it will be more likely to diagnose the presence of fluid in its nascent stages, meaning treatment can be introduced early and before it causes limitations to vision. FD-OCT can also capture macular hemorrhaging. While TD-OCT would also theoretically show fluid and blood at the macula, it would not be able to depict with the same accuracy at what retinal layer the buildup occurs.

Fig. 3. Age-related macular degeneration: a) ocular fundus; b) 2D scan; c) 3D scan (courtesy of Dr. F. Balta, Eye Hospital, Bucharest, Romania)

#### **2.4.2 Cirrus High Definition-OCT (HD-OCT) in the diagnosis of AMD**

Cirrus HD-OCT (Carl Zeiss Meditec) provides detailed maps and quantifies the retinal thickness and volume.The 5-line raster, with over 4.000 A-scans per line, offers such high definition images that differences in fluid are identifiable, thus helping to indicate the specific disease process. The representation of the macula is useful for layer identification, segmentation, and the quantitative and qualitative analysis.

In fig. 3a, the ophthalmoscopic aspect of a wet form of AMD is illustrated: subfoveal neovascular membrane, elevating the retina, on average 1,5 disc diameters in surface. Fig. 3b depicts the 2D FD-OCT image of the macula in wet AMD: elevation of the macular retina, the space under the neurosensory retina is occupied by an irregular, opaque structure; the retinal pigmented epithelium is thickened. The 3D FD-OCT image of the macula in wet AMD (fig. 3c) offers a tridimensional view: elevation of the macular retina, better depiction of the retinal topography, thus improving the visualization of RPE irregularities. The advantage of FD-OCT in AMD is revealed by the fact that because it more densely samples the macula, it will be more likely to diagnose the presence of fluid in its nascent stages, meaning treatment can be introduced early and before it causes limitations to vision. FD-OCT can also capture macular hemorrhaging. While TD-OCT would also theoretically show fluid and blood at the macula, it would not be able to depict with the same accuracy at what

Fig. 3. Age-related macular degeneration: a) ocular fundus; b) 2D scan; c) 3D scan (courtesy

Cirrus HD-OCT (Carl Zeiss Meditec) provides detailed maps and quantifies the retinal thickness and volume.The 5-line raster, with over 4.000 A-scans per line, offers such high definition images that differences in fluid are identifiable, thus helping to indicate the specific disease process. The representation of the macula is useful for layer identification,

**2.4.2 Cirrus High Definition-OCT (HD-OCT) in the diagnosis of AMD** 

segmentation, and the quantitative and qualitative analysis.

of Dr. F. Balta, Eye Hospital, Bucharest, Romania)

retinal layer the buildup occurs.

RPE detachment is characterized by the accumulation of fluid between the highly reflective RPE (red line) and the moderately reflective choriocapillaris. Blister-like elevation of the retina and RPE are evident on the OCT image. Figures 4 and 5 depict the PEDs: the red line representing the RPE is irregular and present focal elevations. Fig. 4 also shows the fluid accumulation in the retina in a region adjacent to the RPE detachment.

Fig. 4. HD-5 line raster: Drusen and PEDs (courtesy of Dr. H. Shah, Midland, Texas, USA)

Occult CNV is a term given to a specific "blothcy" appearance on the angiogram. The occult CNV in fig. 6 is suggested by the irregularities and thickening of the RPE layer and the CME appears like small, cystic spaces within the macula. In the figure above, the occult CNV is not accompanied by PED, therefore it is cathegorized as type 2 occult CNV. The type 1 occult CNV associates PEDs and is also named vascularized PED. The occult CNV might be an early phase of the classic CNV and therefore its identification is important for the early diagnosis of the wet AMD.

When the choroidal new vessels that have grown under the macula are seen on the angiogram, this condition is named classic CNV. The classic CNV appears on OCT as a hyper-reflective structure that elevates the RPE. This aspect is easily demonstrated in fig. 7, 8, and 9.

Use of OCT Imaging in the

Diagnosis and Monitoring of Age Related Macular Degeneration 265

Fig. 7. HD-5 line raster: Classic CNV (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 8. Classic CNV (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 5. HD-5 line raster: PEDs (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 6. Occult CNV with CME (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 5. HD-5 line raster: PEDs (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 6. Occult CNV with CME (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 7. HD-5 line raster: Classic CNV (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 8. Classic CNV (courtesy of Dr. H. Shah, Midland, Texas, USA)

Use of OCT Imaging in the

layers.

Diagnosis and Monitoring of Age Related Macular Degeneration 267

In fig. 10 the RPE appears irregular and thickened (suggesting an occult CNV) and under the RPE line there is a hyper-reflective structure slightly elevating the RPE on line 2 of the raster (classic CNV). On the same line, the RPE line appears interrupted. The structure of the neurosensory retina is disorganized by the cystic fluid accumulation within the retinal

Fig. 11. Choroidal atrophy (courtesy of Dr. H. Shah, Midland, Texas, USA)

autofluorescence, but this hasn't been proved yet.

**2.4.3 TD-OCT (Stratus) in monitoring AMD** 

captures 1 image at a time

Geographic atrophy is the end stage of the dry AMD. The reflectivity of the RPE and underlying choroid is increased (fig. 11). There have been defined spectral-domain OCT patterns that are correlated with a higher risk of atrophy's extension: the irregular margin of the lesion on OCT is significantly associated with increased fundus autofluorescence and a higher risk of progression. The smooth margin of the lesion has been significantly associated with normal fundus autofluorescence and a lower risk of atrophy's progression (Brar M. et al., 2009). In our example (fig. 11), the margins of the choroidal atrophy appear pretty irregular. We can speculate that because it reveals structural changes, the spectral – domain OCT is a better predictor for geographic atrophy's extension, as compared to fundus

The TD-OCT imaging was performed using a Stratus OCT 2006 commercial instrument (Carl Zeiss Meditec, Dublin, California, USA). The Stratus TD-OCT provides real-time crosssectional images and quantitative analysis of retinal features to optimize the diagnosis and monitoring of retinal disease and for enhanced pre-and post-therapy assessment. Sensor

Fig. 9. Classic CNV (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 10. HD-5 line raster: Wet AMD (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 9. Classic CNV (courtesy of Dr. H. Shah, Midland, Texas, USA)

Fig. 10. HD-5 line raster: Wet AMD (courtesy of Dr. H. Shah, Midland, Texas, USA)

In fig. 10 the RPE appears irregular and thickened (suggesting an occult CNV) and under the RPE line there is a hyper-reflective structure slightly elevating the RPE on line 2 of the raster (classic CNV). On the same line, the RPE line appears interrupted. The structure of the neurosensory retina is disorganized by the cystic fluid accumulation within the retinal layers.

Fig. 11. Choroidal atrophy (courtesy of Dr. H. Shah, Midland, Texas, USA)

Geographic atrophy is the end stage of the dry AMD. The reflectivity of the RPE and underlying choroid is increased (fig. 11). There have been defined spectral-domain OCT patterns that are correlated with a higher risk of atrophy's extension: the irregular margin of the lesion on OCT is significantly associated with increased fundus autofluorescence and a higher risk of progression. The smooth margin of the lesion has been significantly associated with normal fundus autofluorescence and a lower risk of atrophy's progression (Brar M. et al., 2009). In our example (fig. 11), the margins of the choroidal atrophy appear pretty irregular. We can speculate that because it reveals structural changes, the spectral – domain OCT is a better predictor for geographic atrophy's extension, as compared to fundus autofluorescence, but this hasn't been proved yet.

#### **2.4.3 TD-OCT (Stratus) in monitoring AMD**

The TD-OCT imaging was performed using a Stratus OCT 2006 commercial instrument (Carl Zeiss Meditec, Dublin, California, USA). The Stratus TD-OCT provides real-time crosssectional images and quantitative analysis of retinal features to optimize the diagnosis and monitoring of retinal disease and for enhanced pre-and post-therapy assessment. Sensor captures 1 image at a time

Use of OCT Imaging in the

injections.

from performing the anti-VEGF intravitreal injections.

Diagnosis and Monitoring of Age Related Macular Degeneration 269

The next case (figure 13) showed a slight improvement in the right eye, 3 months after 3 Bevacizumab injections. The fibrotic nature of the submacular tissue in the left eye kept us

Fig. 13. RTM: comparation of the macular thickness before and 3 months after Bevacizumab

Figure 14 illustrates the decrease of the macular thickness 3 months after 3 Bevacizumab

In all the cases with favorable outcome, the most spectacular improvement, both in vision and in the anatomical aspect, has been obtained after the first injection. The better results are directly correlated with the early stage of the disease. Even if the vision has not changed

injection (courtesy of dr. H. Demea, Review Centre, Cluj-Napoca, Romania)

In figure 12, the comparation of the macular thickness before and 3 months after Bevacizumab injection revealed no significant modification, which led us to stop the anti-VEGF intravitreal injections.

Fig. 12. RTM: comparation of the macular thickness before and 3 months after Bevacizumab injection (courtesy of dr. H. Demea, Review Centre, Cluj-Napoca, Romania)

In figure 12, the comparation of the macular thickness before and 3 months after Bevacizumab injection revealed no significant modification, which led us to stop the anti-

Fig. 12. RTM: comparation of the macular thickness before and 3 months after Bevacizumab

injection (courtesy of dr. H. Demea, Review Centre, Cluj-Napoca, Romania)

VEGF intravitreal injections.

The next case (figure 13) showed a slight improvement in the right eye, 3 months after 3 Bevacizumab injections. The fibrotic nature of the submacular tissue in the left eye kept us from performing the anti-VEGF intravitreal injections.

Fig. 13. RTM: comparation of the macular thickness before and 3 months after Bevacizumab injection (courtesy of dr. H. Demea, Review Centre, Cluj-Napoca, Romania)

Figure 14 illustrates the decrease of the macular thickness 3 months after 3 Bevacizumab injections.

In all the cases with favorable outcome, the most spectacular improvement, both in vision and in the anatomical aspect, has been obtained after the first injection. The better results are directly correlated with the early stage of the disease. Even if the vision has not changed

Use of OCT Imaging in the

**4. Acknowledgment** 

**5. References** 

1, code ID\_459, 2007 - 2010.ID\_459.

**3. Conclusion** 

Diagnosis and Monitoring of Age Related Macular Degeneration 271

The invention of OCT in the 1990s is a major breakthrough in ocular imaging, as it represents a fast, non-invasive, radiation-free examination technique, able to visualize precisely the retinal layers – something done before only on pathology slides. The information offered by OCT is detailed, simple and easily interpretable. AMD is by far, the ocular condition that has benefited the most from the enormous advantages offered by OCT, in terms of diagnosis, response to treatment and monitoring. OCT is applied by two main methods: Time domain OCT (TD-OCT) and Spectral domain OCT (SD-OCT). TD-OCT produces two-dimensional images of the sample internal structure. SD-OCT can be implemented in two formats, Fourier domain (FD-OCT) and swept source (SS-OCT). The image is acquired rapidly - about 60 times faster than with TD-OCT. The image resolution in SD-OCT is 1 μm, as compared to 10 μm in TD-OCT. The practical impact of this improvement in resolution is the early detection of small cystic changes associated with the wet form of AMD. The great number of scans done per second allows SD-OCT systems to generate 3D reconstructions, which can be further manipulated. Visualization of these data in 3D demonstrates subtle pathology that are not evident with conventional 2D images. The most frequent application of OCT in retinal disease is the measurement and monitoring of the retinal thickness. The TD - OCT system (Stratus) gives a macular thickness map which is calculated from 6 radial B-scans crossing at the fovea. By interpolating data from these scans, the average macular thickness is calculated in 9 subfields centered on the fovea. Similarly, the total macular volume is obtained. The SD-OCT provides images with much higher resolution. Given the differences between the measurements with the two types of OCT (TD and SD), algorithms are necessary in order to establish correlations between them. Besides its contribution in the diagnosis of AMD, OCT examination is extremely useful in establishing the indication of the modern treatments in wet AMD (intravitreal injections with anti-VEGF agents) and in the monitoring of the response to treatment. Ideally, the OCT scan must be interpreted in correlation with the fundus photography, direction of scan and ideally, the angiography. Despite the obvious advantages offered by OCT at the present

moment, there is still room for future developments and improvement.

This work has been financially supported by the Romanian Ministry of Education, Research and Youth, through The National University Research Council, Grant PN–II–ID–PCE–2007–

Brar, M.; Kozak, I. & Chang, L. (2009). Correlation between spectral-domain optical

Vol. 23, No 11 (September 2009), pp. 2071 – 2078, ISSN 1552 - 5783

coherence tomography and fundus autofluorescence at the margins of geographic atrophy. *Am J Ophthalmol,*Vol. 148, No 3 (Sept. 2009), pp. 439-444, ISSN 0002-9394 Coscas, G. (2009). *Optical Coherence Tomography in Age-Related Macular Degeneration* (edition 2009), Springer Medizin Verlag, ISBN 978-3-642-01468-0, Heidelberg Forte, R.; Cennamo, CL. & Finelli ML. (2009). Comparison of time domain Stratus OCT and

spectral domain SLO/OCT for assessment of macular thickness and volume. *Eye,* 

from the quantitative point of view, all the patients with a better anatomical aspect of the macula on OCT have also experienced a significant improvement in the quality of vision, translated by the diminishing of the central scotoma, both in surface and in density (Talu et al., 2010).

Fig. 14. RTM: comparison of the macular thickness before and 3 months after Bevacizumab injection (courtesy of dr. H. Demea, Review Centre, Cluj-Napoca, Romania)

#### **3. Conclusion**

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

from the quantitative point of view, all the patients with a better anatomical aspect of the macula on OCT have also experienced a significant improvement in the quality of vision, translated by the diminishing of the central scotoma, both in surface and in density (Talu et

Fig. 14. RTM: comparison of the macular thickness before and 3 months after Bevacizumab

injection (courtesy of dr. H. Demea, Review Centre, Cluj-Napoca, Romania)

al., 2010).

The invention of OCT in the 1990s is a major breakthrough in ocular imaging, as it represents a fast, non-invasive, radiation-free examination technique, able to visualize precisely the retinal layers – something done before only on pathology slides. The information offered by OCT is detailed, simple and easily interpretable. AMD is by far, the ocular condition that has benefited the most from the enormous advantages offered by OCT, in terms of diagnosis, response to treatment and monitoring. OCT is applied by two main methods: Time domain OCT (TD-OCT) and Spectral domain OCT (SD-OCT). TD-OCT produces two-dimensional images of the sample internal structure. SD-OCT can be implemented in two formats, Fourier domain (FD-OCT) and swept source (SS-OCT). The image is acquired rapidly - about 60 times faster than with TD-OCT. The image resolution in SD-OCT is 1 μm, as compared to 10 μm in TD-OCT. The practical impact of this improvement in resolution is the early detection of small cystic changes associated with the wet form of AMD. The great number of scans done per second allows SD-OCT systems to generate 3D reconstructions, which can be further manipulated. Visualization of these data in 3D demonstrates subtle pathology that are not evident with conventional 2D images. The most frequent application of OCT in retinal disease is the measurement and monitoring of the retinal thickness. The TD - OCT system (Stratus) gives a macular thickness map which is calculated from 6 radial B-scans crossing at the fovea. By interpolating data from these scans, the average macular thickness is calculated in 9 subfields centered on the fovea. Similarly, the total macular volume is obtained. The SD-OCT provides images with much higher resolution. Given the differences between the measurements with the two types of OCT (TD and SD), algorithms are necessary in order to establish correlations between them. Besides its contribution in the diagnosis of AMD, OCT examination is extremely useful in establishing the indication of the modern treatments in wet AMD (intravitreal injections with anti-VEGF agents) and in the monitoring of the response to treatment. Ideally, the OCT scan must be interpreted in correlation with the fundus photography, direction of scan and ideally, the angiography. Despite the obvious advantages offered by OCT at the present moment, there is still room for future developments and improvement.

#### **4. Acknowledgment**

This work has been financially supported by the Romanian Ministry of Education, Research and Youth, through The National University Research Council, Grant PN–II–ID–PCE–2007– 1, code ID\_459, 2007 - 2010.ID\_459.

#### **5. References**


Grover, S.; Murthy, RK & Brar VS (2010). Comparison of Retinal Thickness in Normal Eyes Using Stratus and Spectralis Optical Coherence Tomography. *Invest Ophthalmol Vis Sci,* Vol. 51, No 5 (May 2010), pp. 2644-2647, ISSN 0146-0404

**14** 

*USA* 

**Treatments of Dry AMD** 

Age-related macular degeneration (AMD) is the most common cause of legal blindness among those over 65 years of age in the United States (Mitchell et al., 1995; Klein et al., 1992). It is also a debilitating disease on central vision in patients over 50 years old (Ambati et al., 2003). As the baby boom generation ages, the incidence of AMD is expected to triple by the year 2025. It was first described in the medical literature as symmetrical central choroidoretinal disease occurring in senile persons (Hutchison 1875). It was not until 1980 that AMD was regenerated to be a significant cause of blindness in the United States (Leibowitz et al., 1980). Even though the prevalence of AMD is highest among Caucasians in western countries, Asians are as high as Caucasians in the development of AMD (Wang et al, 2010). In 2004, WHO estimated that there are 14 million persons worldwide suffering from blindness or severely impaired vision because of AMD. As the population in the Western World is growing older, the morbidity of losing the ability to read and drive resulting from AMD is becoming increasingly apparent (Klein, 1997). A 2004 analysis reported that among Americans over the age of 40, AMD and/or geographic atrophy were present in at least one eye in 1.47% of the population (Friedman et al., 2004). By the year 2020, there may be a 50% increase in the incidence of AMD. The study predicted that as a result of the rising prevalence of AMD, the number of blind people in the U.S. could increase by as much as 70% by 2020 (Congdon et al., 2004). Because of the enormous impact of AMD on the aging population, much public attention and research has been focused on

The therapy that could treat patients at the dry AMD stage and prevent its progression have a huge impact in reducing the incidence of blindness, improving the quality of life and reducing the social costs of AMD, which equals to approximately \$30 B in GDP annually in

AMD occurs initially in a "dry" form with pathological changes in the retinal pigment epithelium (RPE) and drusen accumulation and can progress to geographic atrophy (GA) (90%) and/or "wet" form of AMD (10%) with choroidal neovascularization (CNV) (Klein, 1997). The breakdown of Bruch's membrane under RPE serves as an entrance for new and immature choroid vessels to grow into the subretinal space that leads to the formation of

**1. Introduction** 

this condition in the past decade.

the year 2003.

George C. Y. Chiou

*College of Medicine* 

*College Station, TX* 

*Institute of Ocular Pharmacology* 

*Texas A&M Health Science Center* 


### **Treatments of Dry AMD**

#### George C. Y. Chiou

*Institute of Ocular Pharmacology College of Medicine Texas A&M Health Science Center College Station, TX USA* 

#### **1. Introduction**

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

Grover, S.; Murthy, RK & Brar VS (2010). Comparison of Retinal Thickness in Normal Eyes

Kakinoki, M.; Sawada, O. & Sawada, T. (2009). Comparison of Macular Thickness Between

Khaderi, K.; Ahmed, K.A. & Berry, G.L. (2011). Retinal Imaging Modalities: Advantages and

Luviano, D.; Benz, M.; & Kim, R. (2009). Selected Clinical Comparisons of Spectral Domain

Marschall, S.; Sander, B. & Mogensen, M. (2011). Optical coherence tomography – current

Mylonas, G.; Ahlers, C. & Malamos, P. (2009). Comparison of retinal thickness

Sayanagi, K.; Sharma, S. & Yamamoto, T. (2009). Comparison of Spectral-Domain versus

Schutze, C.; Ahlers, C. & Sacu, S. (2011). Performance of OCT segmentation procedures to

Talu, S.; Balta, F. & Talu, S.D. (2009). Fourier-Domain Optical Coherence Tomography in

Talu, S.; Demea, H. & Demea, S. (2010). Avastin in age-related macular degeneration, *Oftalmologia,* Vol.54, No 1 (January-March 2010), pp. 95 – 100, ISSN 1220-0875 Walther, J.; Gaertner, M. & Cimalla, P. (2011). Optical coherence tomography in biomedical

Wenner, J.; Wismann, S. & Jäger, M. (2011) Interchangeability of macular thickness

*Sci,* Vol. 51, No 5 (May 2010), pp. 2644-2647, ISSN 0146-0404

Vol. 40, No 3 (May/June 2009), pp. 325-328, ISSN 1542-8877

Vol. 400, No 9 ( May 2011), pp. 2699 – 2720, ISSN 1618 - 2650

Vol. 17, No 1 (January 2011), pp. 15-20, ISSN 1007-9327

89 No 3 (May 2011), pp. 235-240, ISSN 1755-3768

(September 2009), pp. 261-266, ISSN 1680-0737

No 8 (August 2011), pp. 1137-1145, ISSN 0721-832X

2009), pp. 947-955, ISSN 0161-6420

2650

(March/April 2009), pp 135-140, ISSN 1542-8877

48, ISSN 1552-812X

Using Stratus and Spectralis Optical Coherence Tomography. *Invest Ophthalmol Vis* 

Cirrus HD-OCT ans Stratus OCT. *Ophthalmic Surg Lasers Imaging,* Vol. 40, No 2

Limitations for Clinical Practice. *Retinal Physician,* Vol. 8, No 3 (April 2011), pp. 44-

and Time Domain Optical Coherence Tomography. *Ophthalmic Surg Lasers Imaging,* 

technology and applications in clinical and biomedical research. *Anal Bioanal Chem,* 

measurements and segmentation performance of four different spectral and time domain OCT devices in neovascular age-related macular degeneration. *Brit J Ophthalmol,* Vol.93, No 11 (November 2009), pp. 1453 – 1460, ISSN 1468-2079 Osiac, E.; Saftoiu, A. & Gheonea, D.I. (2011). Optical coherence tomography and Doppler

optical coherence tomography in the gastrointestinal tract. *World J Gastroenterol,* 

Time-Domain Optical Coherence Tomography in the Management of Age-Related Macular Degeneration with Ranibizumab, *Ophthalmology,* Vol. 116 No 5 (May

assess morphology and extension in geographic atrophy, *Acta Ophthalmologica,* Vol.

diagnosing and monitoring of retinal diseases. *IFMBE Proceedings,* Vol. 26, No 1

research. *Anal Bioanal Chem,* Vol. 400, No 9 (May 2011), pp. 2721-2743, ISSN 1618-

measurements between different volumetric protocols of Spectralis optical coherence tomography in normal eyes, *Graefes Arch Clin Exp Ophthalmol,* Vol. 249, Age-related macular degeneration (AMD) is the most common cause of legal blindness among those over 65 years of age in the United States (Mitchell et al., 1995; Klein et al., 1992). It is also a debilitating disease on central vision in patients over 50 years old (Ambati et al., 2003). As the baby boom generation ages, the incidence of AMD is expected to triple by the year 2025. It was first described in the medical literature as symmetrical central choroidoretinal disease occurring in senile persons (Hutchison 1875). It was not until 1980 that AMD was regenerated to be a significant cause of blindness in the United States (Leibowitz et al., 1980). Even though the prevalence of AMD is highest among Caucasians in western countries, Asians are as high as Caucasians in the development of AMD (Wang et al, 2010). In 2004, WHO estimated that there are 14 million persons worldwide suffering from blindness or severely impaired vision because of AMD. As the population in the Western World is growing older, the morbidity of losing the ability to read and drive resulting from AMD is becoming increasingly apparent (Klein, 1997). A 2004 analysis reported that among Americans over the age of 40, AMD and/or geographic atrophy were present in at least one eye in 1.47% of the population (Friedman et al., 2004). By the year 2020, there may be a 50% increase in the incidence of AMD. The study predicted that as a result of the rising prevalence of AMD, the number of blind people in the U.S. could increase by as much as 70% by 2020 (Congdon et al., 2004). Because of the enormous impact of AMD on the aging population, much public attention and research has been focused on this condition in the past decade.

The therapy that could treat patients at the dry AMD stage and prevent its progression have a huge impact in reducing the incidence of blindness, improving the quality of life and reducing the social costs of AMD, which equals to approximately \$30 B in GDP annually in the year 2003.

AMD occurs initially in a "dry" form with pathological changes in the retinal pigment epithelium (RPE) and drusen accumulation and can progress to geographic atrophy (GA) (90%) and/or "wet" form of AMD (10%) with choroidal neovascularization (CNV) (Klein, 1997). The breakdown of Bruch's membrane under RPE serves as an entrance for new and immature choroid vessels to grow into the subretinal space that leads to the formation of

Treatments of Dry AMD 275

VEGF has a high degree of selectivity to endothelial cells, reciprocal oxygen regulation, diffusible to its target through extracellular secretion, and affecting multiple components of angiogenesis (endothelial cell proliferation, survival and migration) as well as vascular permeability (Ambati et al., 2003b). There is a lot of evidence showing a putative role of VEGF in CNV formation. Intravitreous injection of an anti-VEGF pegylated aptamer, a synthetic RNA compound specifically designed to bind to extracellular VEGF, stabilized or improved vision in 87.5% of patients with subfoveal CNV 3mo after treatment. However, elimination of VEGF threatens the normal survival of choriocapillaries, which is the trigger of the AMD to begin with. Thus, VEGF inhibitors are double blade swords, which make the control of VEGF levels during the treatment of AMD rather difficult. Inflammation and compromised immune systems are also implicated in the pathogenesis of dry AMD. As a result, anti-inflammatory agents, such as steroids, are frequently tried for the treatment of dry AMD. More specifically, complement components such as C3 and C5 are constituents of drusen in AMD patients (Ambati et al., 2003a). Others, such as interleukin-1, interleukin-6, and tumor necrosis factor (TNF) are implicated to develop dry AMD as well. Thus,

interleukin-1 blockers have been tried in the dry AMD animal models as well.

enzymatic degradation of ECM components.

metalloproteinases (TIMPs) (Ambati, et al., 2003b).

The anti-angiogenic effect of corticosteroids has a dual mechanism. Not only do corticosteroids inhibit inflammation, but they also affect vascular endothelial cell extracellular matrix (ECM) turnover (Kaven, et al., 2001; Danis, et al., 2000). Similarly, corticosteroids decrease RPE cellular migration and proliferation by effecting a diminished

Invasion and migration of endothelial cells through the extracellular matrix during angiogenesis are orchestrated by the integrin family of cell adhesion molecules. They facilitate migration by interacting with adhesion proteins in the ECM, such as collagen, fibronectin, fibrinogen, laminin, vitronectin and von Willebrand factor. The process of interacting with adhesion proteins was potentiated by the secretion of matrix metalloproteinases (MMPs), a family of proteolytic enzymes that degrade basement membrane and extracellular matrix proteins, modulated by tissue inhibitors of

Drugs, which can change the construction of ECM or change the balance of MMPs and TIMPs, may have effects on angiogenesis process. Integrin alpha(v) beta3 is predominately expressed on endothelial cells in choroidal neovascularization (CNV). N-Biphenyl sulfonylphenylalanine hydroxamic acid (BPHA) is a synthetic, selective inhibitor of matrix metalloproteinase (MMP)-2, -9, -14. Oral administration of BPHA can reduce experimental laser-induced choroidal neovascularization (Kohri, et al., 2003). The binding of urokinase plasminogen activator (uPA) and its receptor (uPAR) triggers twin cascades of events during cancer research, the first of which is destruction of the extracellular matrix, and the second is intracellular signaling to program gene expression leading to cell migration, cell invasion, metasis, and angiogenesis. Overexpression of uPA/uPAR system has been shown in surgically excised CNV, and in laser-induced CNV. The octapeptide A6 is derived from the non-receptor-binding regulation of uPA. Subretinal injection of adenoviral or adenoassociated viral vectors have been used to transform the RPE into a factory for sustained local delivery of a drug or gene in experimental models of CNV. Angiostatin (act as a VEGF scavenger), TIMP-3. PEDF has been tested and showed inhibition of development of CNV in animal models (Ambati, et al., 2003b). An antiangiogenic activity that may last for several

CNV (Klein, 1997; Lin and Chiou, 2008; Algveve and Seregard, 2002; Joussen, 2004). CNV can leak fluid as well as hemorrhage in the subretinal space resulting in blurry vision, visual distortion and sudden loss of vision (Nowak, 2006). If left untreated, these lesions progress to form an organized fibrous scar, termed diciform scar, which results in irreversible central vision loss.

The precise etiology is poorly understood despite intensive researches. Thus, we have limited choices of treatment for this kind of disease. Available treatment can be grouped into two major categories: physical and pharmacological (chemical) therapies. The former received extensive attention with little success whereas the latter attract new attention with great hope of success.

### **2. Etiology and pathogenesis of dry AMD**

Drusen is a typical clinicopathologic entity in dry AMD, which causes the changes of retinal pigment epithelium (RPE) and Bruch's membrane (BRM). Drusen is deposited in between the basement membrane of RPE and BRM or external to BRM (Hope et al., 1992). The prevalence and severity of drusen formation in the eyes are linearly related to the progression of AMD. Oxydative stress has long been linked to age-related degenerative diseases and is implicated in the pathogenesis of AMD. The oxidative damage is likely to be the photo reactive pigments which accumulate progressively and constitute the lipofuscin of RPE cells (Imamura et al., 2006; Zhou et al, 2006).

The two major carotenoids in the human macula and retina are lutein and zeaxanthin. Lutein and zeaxanthin are deposited at an up to 5 fold higher content in the macular region of the retina as compared to the peripheral retina. Several functions of these pigments have been hypothesized and these include limitation of the damaging photo-oxidative effects of blue light through its absorption, reduction of the effects of light scatter and chromatic aberration on visual performance, and protection against the adverse effects of photochemical reactions because of the anti-oxidant properties of the carotenoids. So it has been further hypothesized that dietary supplementation with lutein and/or zeaxanthin might protect the retina and/or delay the progression of AMD (Mozaffarieh et al., 2003). Data from the Age-Related Eye Disease Study (AREDS) suggests that supplements that contain carotenoids, anti-oxidant vitamins A, C, and E, and minerals, such as zinc, showed a 25% decrease in the rate of progression to aggressive AMD among high risk patients (AREDS Report, 2001). The findings of the Lutein Anti-oxidant Supplementation Trial (LAST), a prospective, 12-month, randomized, double-masked, placebo-controlled trial, also support a possible therapeutic role of lutein in AMD (Richer et al., 2004). However, the controversial evidence also exists. The information available provides an indication that the carotenoids, lutein and zeaxanthin, may play a role in modulating the course of AMD, yet critical evidence of the beneficial effect has not been found, and crucial information for the most effective design of clinical trials is needed.

Similar to drusen, basal laminar deposit (BLD) is another typical sign of AMD development and led by extracellular deposit. BLD is located between the cell membrane of RPE and its basement membrane (Green and Enger, 1993; Kliffen et al., 1997). The pathogenesis of BLD could be enhanced by a high fat/cholesterol (HFC) diet. The accumulation of lipid particles in BRM is often associated with vascular endothelial growth factor (VEGF) expression and eventual development of CNV in wet AMD (Rudolf et al., 2005).

CNV (Klein, 1997; Lin and Chiou, 2008; Algveve and Seregard, 2002; Joussen, 2004). CNV can leak fluid as well as hemorrhage in the subretinal space resulting in blurry vision, visual distortion and sudden loss of vision (Nowak, 2006). If left untreated, these lesions progress to form an organized fibrous scar, termed diciform scar, which results in irreversible central

The precise etiology is poorly understood despite intensive researches. Thus, we have limited choices of treatment for this kind of disease. Available treatment can be grouped into two major categories: physical and pharmacological (chemical) therapies. The former received extensive attention with little success whereas the latter attract new attention with

Drusen is a typical clinicopathologic entity in dry AMD, which causes the changes of retinal pigment epithelium (RPE) and Bruch's membrane (BRM). Drusen is deposited in between the basement membrane of RPE and BRM or external to BRM (Hope et al., 1992). The prevalence and severity of drusen formation in the eyes are linearly related to the progression of AMD. Oxydative stress has long been linked to age-related degenerative diseases and is implicated in the pathogenesis of AMD. The oxidative damage is likely to be the photo reactive pigments which accumulate progressively and constitute the lipofuscin of

The two major carotenoids in the human macula and retina are lutein and zeaxanthin. Lutein and zeaxanthin are deposited at an up to 5 fold higher content in the macular region of the retina as compared to the peripheral retina. Several functions of these pigments have been hypothesized and these include limitation of the damaging photo-oxidative effects of blue light through its absorption, reduction of the effects of light scatter and chromatic aberration on visual performance, and protection against the adverse effects of photochemical reactions because of the anti-oxidant properties of the carotenoids. So it has been further hypothesized that dietary supplementation with lutein and/or zeaxanthin might protect the retina and/or delay the progression of AMD (Mozaffarieh et al., 2003). Data from the Age-Related Eye Disease Study (AREDS) suggests that supplements that contain carotenoids, anti-oxidant vitamins A, C, and E, and minerals, such as zinc, showed a 25% decrease in the rate of progression to aggressive AMD among high risk patients (AREDS Report, 2001). The findings of the Lutein Anti-oxidant Supplementation Trial (LAST), a prospective, 12-month, randomized, double-masked, placebo-controlled trial, also support a possible therapeutic role of lutein in AMD (Richer et al., 2004). However, the controversial evidence also exists. The information available provides an indication that the carotenoids, lutein and zeaxanthin, may play a role in modulating the course of AMD, yet critical evidence of the beneficial effect has not been found, and crucial information for the

Similar to drusen, basal laminar deposit (BLD) is another typical sign of AMD development and led by extracellular deposit. BLD is located between the cell membrane of RPE and its basement membrane (Green and Enger, 1993; Kliffen et al., 1997). The pathogenesis of BLD could be enhanced by a high fat/cholesterol (HFC) diet. The accumulation of lipid particles in BRM is often associated with vascular endothelial growth factor (VEGF) expression and

vision loss.

great hope of success.

**2. Etiology and pathogenesis of dry AMD** 

RPE cells (Imamura et al., 2006; Zhou et al, 2006).

most effective design of clinical trials is needed.

eventual development of CNV in wet AMD (Rudolf et al., 2005).

VEGF has a high degree of selectivity to endothelial cells, reciprocal oxygen regulation, diffusible to its target through extracellular secretion, and affecting multiple components of angiogenesis (endothelial cell proliferation, survival and migration) as well as vascular permeability (Ambati et al., 2003b). There is a lot of evidence showing a putative role of VEGF in CNV formation. Intravitreous injection of an anti-VEGF pegylated aptamer, a synthetic RNA compound specifically designed to bind to extracellular VEGF, stabilized or improved vision in 87.5% of patients with subfoveal CNV 3mo after treatment. However, elimination of VEGF threatens the normal survival of choriocapillaries, which is the trigger of the AMD to begin with. Thus, VEGF inhibitors are double blade swords, which make the control of VEGF levels during the treatment of AMD rather difficult. Inflammation and compromised immune systems are also implicated in the pathogenesis of dry AMD. As a result, anti-inflammatory agents, such as steroids, are frequently tried for the treatment of dry AMD. More specifically, complement components such as C3 and C5 are constituents of drusen in AMD patients (Ambati et al., 2003a). Others, such as interleukin-1, interleukin-6, and tumor necrosis factor (TNF) are implicated to develop dry AMD as well. Thus, interleukin-1 blockers have been tried in the dry AMD animal models as well.

The anti-angiogenic effect of corticosteroids has a dual mechanism. Not only do corticosteroids inhibit inflammation, but they also affect vascular endothelial cell extracellular matrix (ECM) turnover (Kaven, et al., 2001; Danis, et al., 2000). Similarly, corticosteroids decrease RPE cellular migration and proliferation by effecting a diminished enzymatic degradation of ECM components.

Invasion and migration of endothelial cells through the extracellular matrix during angiogenesis are orchestrated by the integrin family of cell adhesion molecules. They facilitate migration by interacting with adhesion proteins in the ECM, such as collagen, fibronectin, fibrinogen, laminin, vitronectin and von Willebrand factor. The process of interacting with adhesion proteins was potentiated by the secretion of matrix metalloproteinases (MMPs), a family of proteolytic enzymes that degrade basement membrane and extracellular matrix proteins, modulated by tissue inhibitors of metalloproteinases (TIMPs) (Ambati, et al., 2003b).

Drugs, which can change the construction of ECM or change the balance of MMPs and TIMPs, may have effects on angiogenesis process. Integrin alpha(v) beta3 is predominately expressed on endothelial cells in choroidal neovascularization (CNV). N-Biphenyl sulfonylphenylalanine hydroxamic acid (BPHA) is a synthetic, selective inhibitor of matrix metalloproteinase (MMP)-2, -9, -14. Oral administration of BPHA can reduce experimental laser-induced choroidal neovascularization (Kohri, et al., 2003). The binding of urokinase plasminogen activator (uPA) and its receptor (uPAR) triggers twin cascades of events during cancer research, the first of which is destruction of the extracellular matrix, and the second is intracellular signaling to program gene expression leading to cell migration, cell invasion, metasis, and angiogenesis. Overexpression of uPA/uPAR system has been shown in surgically excised CNV, and in laser-induced CNV. The octapeptide A6 is derived from the non-receptor-binding regulation of uPA. Subretinal injection of adenoviral or adenoassociated viral vectors have been used to transform the RPE into a factory for sustained local delivery of a drug or gene in experimental models of CNV. Angiostatin (act as a VEGF scavenger), TIMP-3. PEDF has been tested and showed inhibition of development of CNV in animal models (Ambati, et al., 2003b). An antiangiogenic activity that may last for several

Treatments of Dry AMD 277

choriocapillaries, enhancing leakage and deposition of extracellular proteins and lipids, particularly in the posterior pole. These deposits take the form of basal deposits within Bruch's membrane and of drusen, which can comprise the overlying retinal pigment epithelium and cause geographic atrophy of RPE. The progressive deposition of lipid in Bruch's membrane results in the degeneration of elastin and collagen, and ultimately calcification. The combination of elevated choriocapillary pressure, vascular endothelial growth factor, and a break in a calcified Bruch's membrane causes choroidal neovascularization in the neovascular form of AMD. Drusen, as well as the decrease in choroidal blood flow may be epiphenomena (Friedman, 2000; 2004). Vasoactive agents that selectively decrease postcapillary choroidal resistance may prevent the development of

Treatment of AMD was initially focused in wet AMD and dry AMD was left untreated because no effective method was available then. Drusen is a marker for dry AMD as the size and number of drusen are proportionally related to the progression of the disease. However it was not clear whether it had a role in the pathogenesis of the disease until 2006 (CAPT) when laser therapy was applied to treat more than 1,000 patients in one eye and the other eye serving as the control. It was found that laser therapy had neither beneficial nor harmful effect for these patients, if any. The laser's energy can disrupt Bruch's membrane which loses the ability to prevent the growth of CNV under the retina, thus converting dry AMD to wet AMD (CAPT, 2006). On the other hand, laser therapy seems to delay the development of CNV by 6 months in studies, including patients with unilateral advanced AMD (Owen et al., 2006; Frikerg et al., 2006). In conclusion, presence and/or elimination of drusen with laser treatment did not affect the visual acuity, indicating that dry AMD cannot be treated

Most research and developments of dry AMD are focused on the prevention of metabolic wastes production with limited success. This is mainly because the production of metabolic wastes comes from numerous sources, including oxidation, aging, complement components, cytokines, inflammations, PEDF, VEGF, ECM turnover and the like. Thus, a complete inhibition of one branch of all pathogens can suppress the progression of dry AMD at only around 20% at best, which falls to the borderline efficacy only. Besides, metabolic wastes are normal products of physiological procedures of the body and complete inhibition of normal metabolism could result in other various pathological side effects. Further, visual acuity does NOT change significantly during the progression of dry AMD, thus, selection of proper end points to evaluate drug efficacy in the clinical trials are very difficult if not impossible.

Although fruitful progress has been made in the treatment of wet AMD, the treatment of dry AMD is still in the desert stage. There is no single drug available in the whole world for the treatment of dry AMD. Since 90% of AMD patient population is in the dry stage, there is active research carried out at the present time at different stages of research and

CNV. Drugs working in this field may provide a new way for AMD treatment.

**3. Treatment of dry AMD with physical means** 

with physical means at the present time.

**4.1 Choroidal blood flow facilitators** 

development (Zou and Chiou, 2005).

**4. Treatment of dry AMD with pharmacological agents** 

months after a single intravitreous injection of doses greater than 10(8) pu of AdPEDF. 11 have been reported. This study showed that adenoviral vector-mediated ocular gene transfer is a viable approval for the treatment of neovascular AMD (Rasmussen et.al., 2001; Campochiaro et al., 2006).

In addition to age, high fat diet, light oxidation, and inflammation, the factors of smoke, alcohol, and gene are frequently questioned. Cigarette smoke has been indicated by epidemiologic studies that it is the single greatest environmental risk factor for both dry and wet AMD (Evans, 2001). Mice experiments with inhaled cigarette smoke resulted in the formation of such-RPE deposits, thickening of BRM and accumulation of deposits, within BRM's membrane (Marin-Castano et al., 2006). On another experiment, mice were fed with nicotine in drinking water, the results showed nicotine increased the size and severity of experimental CNV formation (Suner, 2004).

The influence of alcohol on the development of CNV in wet AMD was studied by Bora et al (2006). The results showed that the activity of fatty acid ethyl ester synthase (FAEES) activity increased 4-fold in the choroid of alcohol treated rats as compared to controls. Further, the amount of ethylesters produced in the choroid was 10-fold higher in alcohol fed rats than the controls. The size of the CNV formation induced by laser treatment increased by 28% due to alcohol treatment.

In addition to environmental factors, gene also plays an important role in the development of dry AMD. Thus, some animal models used for AMD studies are transgenic mice treated with blue-green light (Espinosa-Heldmann et al., 2004).

There are some diseases which are similar to AMD. They include Stargardt macular dystrophy (STGD) and Sorsby's fundus dystrophy (SFD). STGD is characterized as dry AMD by accumulation of high level of lipofuscin in the RPE. It precedes to degeneration of photocells in the macular and atrophy of RPE (Karan et al., 2005; Raz-Prag et al., 2006). SFD is a rare autosomal dominant disorder that results in degeneration of the macular region, which leads to rapid loss of central vision like wet AMD (Li et al., 2005).

Most importantly, the choroidal blood flow of dry AMD and STGD is compromised and significantly lower than in normal eyes. (Grunwald et al., 2003, 2005) As a result, all metabolic wastes produced from oxidation, inflammation, aging, complement components, cytokines, cigarette smoke, nicotine, high lipid, alcohol and anything else are accumulated in RPE cells and BRM which trigger dry AMD and eventually wet AMD or GA. On the opposite direction, nutrient supply to BRM, RPE and photo cells at macula are markedly reduced which facilitate the worsening of dry AMD (Jiang and Chiou, 2007).

It is noteworthy that choroidal blood flow is found to be impaired by every method used to quantify it in the aging eye and in age-related macular degeneration: flourescein and indocyanine green angiography, color Doppler imaging, laser Doppler flowmetry, and pulsatile ocular blood flow (Freidman, 2000). The vascular model of AMD suggests that the elevation of intravascular pressure is the crucial hemodynamic factor in age-related macular degeneration. AMD is the result of the accumulation of lipids in the sclera and in Bruch's membrane, progressively increasing the stiffness of these tissues, and increasing the postcapillary resistance of the choroidal vasculature. In addition to decreasing choroidal blood flow, the increase in resistance tends to elevate the hydrostatic pressure of the

months after a single intravitreous injection of doses greater than 10(8) pu of AdPEDF. 11 have been reported. This study showed that adenoviral vector-mediated ocular gene transfer is a viable approval for the treatment of neovascular AMD (Rasmussen et.al., 2001;

In addition to age, high fat diet, light oxidation, and inflammation, the factors of smoke, alcohol, and gene are frequently questioned. Cigarette smoke has been indicated by epidemiologic studies that it is the single greatest environmental risk factor for both dry and wet AMD (Evans, 2001). Mice experiments with inhaled cigarette smoke resulted in the formation of such-RPE deposits, thickening of BRM and accumulation of deposits, within BRM's membrane (Marin-Castano et al., 2006). On another experiment, mice were fed with nicotine in drinking water, the results showed nicotine increased the size and severity of

The influence of alcohol on the development of CNV in wet AMD was studied by Bora et al (2006). The results showed that the activity of fatty acid ethyl ester synthase (FAEES) activity increased 4-fold in the choroid of alcohol treated rats as compared to controls. Further, the amount of ethylesters produced in the choroid was 10-fold higher in alcohol fed rats than the controls. The size of the CNV formation induced by laser treatment increased

In addition to environmental factors, gene also plays an important role in the development of dry AMD. Thus, some animal models used for AMD studies are transgenic mice treated

There are some diseases which are similar to AMD. They include Stargardt macular dystrophy (STGD) and Sorsby's fundus dystrophy (SFD). STGD is characterized as dry AMD by accumulation of high level of lipofuscin in the RPE. It precedes to degeneration of photocells in the macular and atrophy of RPE (Karan et al., 2005; Raz-Prag et al., 2006). SFD is a rare autosomal dominant disorder that results in degeneration of the macular region,

Most importantly, the choroidal blood flow of dry AMD and STGD is compromised and significantly lower than in normal eyes. (Grunwald et al., 2003, 2005) As a result, all metabolic wastes produced from oxidation, inflammation, aging, complement components, cytokines, cigarette smoke, nicotine, high lipid, alcohol and anything else are accumulated in RPE cells and BRM which trigger dry AMD and eventually wet AMD or GA. On the opposite direction, nutrient supply to BRM, RPE and photo cells at macula are markedly

It is noteworthy that choroidal blood flow is found to be impaired by every method used to quantify it in the aging eye and in age-related macular degeneration: flourescein and indocyanine green angiography, color Doppler imaging, laser Doppler flowmetry, and pulsatile ocular blood flow (Freidman, 2000). The vascular model of AMD suggests that the elevation of intravascular pressure is the crucial hemodynamic factor in age-related macular degeneration. AMD is the result of the accumulation of lipids in the sclera and in Bruch's membrane, progressively increasing the stiffness of these tissues, and increasing the postcapillary resistance of the choroidal vasculature. In addition to decreasing choroidal blood flow, the increase in resistance tends to elevate the hydrostatic pressure of the

Campochiaro et al., 2006).

experimental CNV formation (Suner, 2004).

with blue-green light (Espinosa-Heldmann et al., 2004).

which leads to rapid loss of central vision like wet AMD (Li et al., 2005).

reduced which facilitate the worsening of dry AMD (Jiang and Chiou, 2007).

by 28% due to alcohol treatment.

choriocapillaries, enhancing leakage and deposition of extracellular proteins and lipids, particularly in the posterior pole. These deposits take the form of basal deposits within Bruch's membrane and of drusen, which can comprise the overlying retinal pigment epithelium and cause geographic atrophy of RPE. The progressive deposition of lipid in Bruch's membrane results in the degeneration of elastin and collagen, and ultimately calcification. The combination of elevated choriocapillary pressure, vascular endothelial growth factor, and a break in a calcified Bruch's membrane causes choroidal neovascularization in the neovascular form of AMD. Drusen, as well as the decrease in choroidal blood flow may be epiphenomena (Friedman, 2000; 2004). Vasoactive agents that selectively decrease postcapillary choroidal resistance may prevent the development of CNV. Drugs working in this field may provide a new way for AMD treatment.

#### **3. Treatment of dry AMD with physical means**

Treatment of AMD was initially focused in wet AMD and dry AMD was left untreated because no effective method was available then. Drusen is a marker for dry AMD as the size and number of drusen are proportionally related to the progression of the disease. However it was not clear whether it had a role in the pathogenesis of the disease until 2006 (CAPT) when laser therapy was applied to treat more than 1,000 patients in one eye and the other eye serving as the control. It was found that laser therapy had neither beneficial nor harmful effect for these patients, if any. The laser's energy can disrupt Bruch's membrane which loses the ability to prevent the growth of CNV under the retina, thus converting dry AMD to wet AMD (CAPT, 2006). On the other hand, laser therapy seems to delay the development of CNV by 6 months in studies, including patients with unilateral advanced AMD (Owen et al., 2006; Frikerg et al., 2006). In conclusion, presence and/or elimination of drusen with laser treatment did not affect the visual acuity, indicating that dry AMD cannot be treated with physical means at the present time.

#### **4. Treatment of dry AMD with pharmacological agents**

Most research and developments of dry AMD are focused on the prevention of metabolic wastes production with limited success. This is mainly because the production of metabolic wastes comes from numerous sources, including oxidation, aging, complement components, cytokines, inflammations, PEDF, VEGF, ECM turnover and the like. Thus, a complete inhibition of one branch of all pathogens can suppress the progression of dry AMD at only around 20% at best, which falls to the borderline efficacy only. Besides, metabolic wastes are normal products of physiological procedures of the body and complete inhibition of normal metabolism could result in other various pathological side effects. Further, visual acuity does NOT change significantly during the progression of dry AMD, thus, selection of proper end points to evaluate drug efficacy in the clinical trials are very difficult if not impossible.

#### **4.1 Choroidal blood flow facilitators**

Although fruitful progress has been made in the treatment of wet AMD, the treatment of dry AMD is still in the desert stage. There is no single drug available in the whole world for the treatment of dry AMD. Since 90% of AMD patient population is in the dry stage, there is active research carried out at the present time at different stages of research and development (Zou and Chiou, 2005).

Treatments of Dry AMD 279

content in the macular region as compared to the peripheral retina. Because of antioxidant properties of carotenoids, lutein and zeaxanthin are considered to be able to protect and/or

Age-related Eye Disease Study (AREDS, 2001) reported that food supplements that contain carotenoids, anti-oxidants vitamins A, C, and E, plus minerals, such as zinc, showed a 25% decrease in the rate of progression to aggressive AMD among high risk patients. The findings of Lutein Anti-oxidant Supplementation Trial (LAST) (Richer et al., 2004) also support a possible therapeutic role of Lutein in AMD treatment. However, the critical

In order to improve the initial success, AREDS-2 has been initiated with a new formulation for a six-year, multicenter, randomized trial. The new formula contains higher doses of lutein and zeaxanthin and/or Omega-3 fatty acids known as DHA and EPA. It also lowers the zinc from 80mg to 25mg and deletes β-carotene. Four thousand participants at ages 50 to 85 have been enrolled and the trial will last six years. The results should come out sometime

All agents, including hydralazine, tetramethylpyrazine, flavone, naringenin, apigenin, quercetin and guanabenz (Cheng and Chiou, 2008; Zou et al., 2006; 2007; Shen et al., 2010a; 2010b; 2011; Zhuang et al., 2010a; 2010b; 2011; Lin and Chiou, 2008) presented in section 4.1 as choroidal blood flow facilitators produce potent anti-oxidating actions as well. They can antagonize oxidation induced injuries on human RPE cells induced by H₂O₂, NaIO3, t-BHP,

OT-551 is a piperidine derivative converted by ocular esterases to the actual metabolite, TEMPOL-H (TP-H) which serves as a potent free radical scavenger. It is a small molecule which can be administered as local eye drops (Tanito et al., 2007), OT-551 also possesses anti-inflammatory and antiangiogenic properties. OT-551 users are being investigated for the therapy of geographic atrophy in AMD. Unfortunately, the phase II trials failed to

Genetic association studies have shown that inflammation appears to be related to AMD (Patel and Chan, 2008). Further, complement factor H (CFH) was associated with an increased risk of developing AMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005). Later studies linked AMD to even more complicated complement system. These studies indicated that inhibition of complement activation would be a reasonable strategy for the treatment of AMD. However, after a life-long damage on complement system, such a strategy might have little benefit on the AMD progression later

Regardless, active investigation was carried out and numerous complement inhibitors were derived for clinical trials. Among them, POT-4 (Potentia Pharmaceuticals) was developed as a C3 inhibitor for wet AMD. Eculizumab (Soliris, Alexion Pharmaceuticals) was developed as a C5 antibody for the treatment of dry AMD. Since eculizumab is a monoclonal antibody, it has to be administered via intravenous infusion to patients with GA or high risk drusen. ARC-1905 (Ophthotech) is an anti-C5 aptamer administrated via intravitreal injection for

produce efficacy in preventing the enlargement rate of GA in AMD (OT-551, 2007).

delay the progression of dry AMD (Mozaffarieh et al., 2003).

evidence of therapeutic efficacy has not been established.

in the near future (Karmel, 2011).

**4.3 Anti-inflammatory agents** 

in life (Arons, 2009).

and the like.

A novel idea to solve the problem has been developed recently by Chiou in Texas A&M Health Science Center. He based on the risk factor of dry AMD as a reduction of choroidal blood flow (Friedman, 2000; Grundwald et al., 2003; 2005; Xu et al., 2010; Figueroa et al., 2006; Metelitsina et al., 2006), which lead to the accumulation of all waste products regardless of where they came from, including aging, oxidation, inflammation, complement components, VEGF, PEDF, cytokines and the like. Thus, instead of solving problems individually by using inhibiting/blocking agents of aging, oxidation, inflammation, complement components, VEGF, PEDF and cytokines, all waste products will be eliminated by improved choroidal circulation. Further, nutrients will be replenished to BRM, RPE, and photocells via improved choroidal circulation in macula to improve the vision.

In order to prove the concept that the disease severity of dry AMD is inversely proportional to choroidal blood flow (CBF), drugs that can facilitate CBF were identified with ocular hypertensive rabbits. The CBF was measured precisely with colored microsphere method. Drugs which can increase CBF were then administered to AMD rat models, including NaIO3 induced and laser-induced AMD models. The former was used to prevent/reverse degeneration of RPE by drugs, representing the treatments of dry AMD. The latter was used to show the prevention of conversion of dry to wet AMD after the Bruch's membrane was broken down by laser treatment. It was interesting to note that those agents which can facilitate CBF can prevent/reverse AMD induced by NaIO3 and/or laser beam. Those agents which did not improve CBF showed no effect on the AMD development. All drugs were administered with eye drops without preservatives in the ophthalmic solution. Phase I clinical trials and proof of the concept of MC1101 (MacuClear) in human patients had been carried out with encouraging results showing no major side effects observed and the drug can reach the back of the eye to facilitate CBF. If preclinical animal data are good indicators, it would most likely show drug efficacy in preventing/reversing the progression of dry AMD with these agents.

Numerous agents that can increase choroidal blood flow in rabbit eyes have been tested in dry AMD animal models. Among them, some were found to be quite efficacious in inhibiting the development of dry AMD. They include, but are not limited to, hydralazine (Jiang and Chiou, 2008; Jiang et al., 2008; Cheng and Chiou, 2008), tetramethylpyrazine (Zou et al., 2007; Shen et al., 2010a), flavone (Zhuang et al., 2010a; 2010b), naringenin (Lin and Chiou, 2008; Shen et al., 2010b), apigenin (Zou and Chiou, 2006), quercetin (Zhuang et al., 2011), guanabenz (Shen et al., 2011), interleukin-1 blockers (Zou et al., 2006) and D-timolol (Xu et al., 2005).

Reduction in choroidal blood flow causes deposition of extracellular proteins, lipids and metabolic wastes in the form of basal deposits within BRM and drusen in between BRM and RPE. The progressive deposition of lipid in BRM results in the degeneration of elastin and collagen, and ultimately calcification. The combination of elevated choriocapillary pressure, expansion of VEGF and break in the calcified BRM causes development of CNV and wet AMD. Vasoactive agents, which can facilitate choroidal blood flow are believed to prevent the progression of dry AMD and is the major focus of the research at the present time (Zou and Chiou, 2005).

#### **4.2 Anti-oxidants**

Antioxidants are the agents most extensively studied. Lutein and zeaxanthin are two major carotenoids in the human macula and retina. They are deposited at an up to 5-fold higher

A novel idea to solve the problem has been developed recently by Chiou in Texas A&M Health Science Center. He based on the risk factor of dry AMD as a reduction of choroidal blood flow (Friedman, 2000; Grundwald et al., 2003; 2005; Xu et al., 2010; Figueroa et al., 2006; Metelitsina et al., 2006), which lead to the accumulation of all waste products regardless of where they came from, including aging, oxidation, inflammation, complement components, VEGF, PEDF, cytokines and the like. Thus, instead of solving problems individually by using inhibiting/blocking agents of aging, oxidation, inflammation, complement components, VEGF, PEDF and cytokines, all waste products will be eliminated by improved choroidal circulation. Further, nutrients will be replenished to BRM, RPE, and

In order to prove the concept that the disease severity of dry AMD is inversely proportional to choroidal blood flow (CBF), drugs that can facilitate CBF were identified with ocular hypertensive rabbits. The CBF was measured precisely with colored microsphere method. Drugs which can increase CBF were then administered to AMD rat models, including NaIO3 induced and laser-induced AMD models. The former was used to prevent/reverse degeneration of RPE by drugs, representing the treatments of dry AMD. The latter was used to show the prevention of conversion of dry to wet AMD after the Bruch's membrane was broken down by laser treatment. It was interesting to note that those agents which can facilitate CBF can prevent/reverse AMD induced by NaIO3 and/or laser beam. Those agents which did not improve CBF showed no effect on the AMD development. All drugs were administered with eye drops without preservatives in the ophthalmic solution. Phase I clinical trials and proof of the concept of MC1101 (MacuClear) in human patients had been carried out with encouraging results showing no major side effects observed and the drug can reach the back of the eye to facilitate CBF. If preclinical animal data are good indicators, it would most likely show drug

photocells via improved choroidal circulation in macula to improve the vision.

efficacy in preventing/reversing the progression of dry AMD with these agents.

(Xu et al., 2005).

and Chiou, 2005).

**4.2 Anti-oxidants** 

Numerous agents that can increase choroidal blood flow in rabbit eyes have been tested in dry AMD animal models. Among them, some were found to be quite efficacious in inhibiting the development of dry AMD. They include, but are not limited to, hydralazine (Jiang and Chiou, 2008; Jiang et al., 2008; Cheng and Chiou, 2008), tetramethylpyrazine (Zou et al., 2007; Shen et al., 2010a), flavone (Zhuang et al., 2010a; 2010b), naringenin (Lin and Chiou, 2008; Shen et al., 2010b), apigenin (Zou and Chiou, 2006), quercetin (Zhuang et al., 2011), guanabenz (Shen et al., 2011), interleukin-1 blockers (Zou et al., 2006) and D-timolol

Reduction in choroidal blood flow causes deposition of extracellular proteins, lipids and metabolic wastes in the form of basal deposits within BRM and drusen in between BRM and RPE. The progressive deposition of lipid in BRM results in the degeneration of elastin and collagen, and ultimately calcification. The combination of elevated choriocapillary pressure, expansion of VEGF and break in the calcified BRM causes development of CNV and wet AMD. Vasoactive agents, which can facilitate choroidal blood flow are believed to prevent the progression of dry AMD and is the major focus of the research at the present time (Zou

Antioxidants are the agents most extensively studied. Lutein and zeaxanthin are two major carotenoids in the human macula and retina. They are deposited at an up to 5-fold higher content in the macular region as compared to the peripheral retina. Because of antioxidant properties of carotenoids, lutein and zeaxanthin are considered to be able to protect and/or delay the progression of dry AMD (Mozaffarieh et al., 2003).

Age-related Eye Disease Study (AREDS, 2001) reported that food supplements that contain carotenoids, anti-oxidants vitamins A, C, and E, plus minerals, such as zinc, showed a 25% decrease in the rate of progression to aggressive AMD among high risk patients. The findings of Lutein Anti-oxidant Supplementation Trial (LAST) (Richer et al., 2004) also support a possible therapeutic role of Lutein in AMD treatment. However, the critical evidence of therapeutic efficacy has not been established.

In order to improve the initial success, AREDS-2 has been initiated with a new formulation for a six-year, multicenter, randomized trial. The new formula contains higher doses of lutein and zeaxanthin and/or Omega-3 fatty acids known as DHA and EPA. It also lowers the zinc from 80mg to 25mg and deletes β-carotene. Four thousand participants at ages 50 to 85 have been enrolled and the trial will last six years. The results should come out sometime in the near future (Karmel, 2011).

All agents, including hydralazine, tetramethylpyrazine, flavone, naringenin, apigenin, quercetin and guanabenz (Cheng and Chiou, 2008; Zou et al., 2006; 2007; Shen et al., 2010a; 2010b; 2011; Zhuang et al., 2010a; 2010b; 2011; Lin and Chiou, 2008) presented in section 4.1 as choroidal blood flow facilitators produce potent anti-oxidating actions as well. They can antagonize oxidation induced injuries on human RPE cells induced by H₂O₂, NaIO3, t-BHP, and the like.

OT-551 is a piperidine derivative converted by ocular esterases to the actual metabolite, TEMPOL-H (TP-H) which serves as a potent free radical scavenger. It is a small molecule which can be administered as local eye drops (Tanito et al., 2007), OT-551 also possesses anti-inflammatory and antiangiogenic properties. OT-551 users are being investigated for the therapy of geographic atrophy in AMD. Unfortunately, the phase II trials failed to produce efficacy in preventing the enlargement rate of GA in AMD (OT-551, 2007).

#### **4.3 Anti-inflammatory agents**

Genetic association studies have shown that inflammation appears to be related to AMD (Patel and Chan, 2008). Further, complement factor H (CFH) was associated with an increased risk of developing AMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005). Later studies linked AMD to even more complicated complement system. These studies indicated that inhibition of complement activation would be a reasonable strategy for the treatment of AMD. However, after a life-long damage on complement system, such a strategy might have little benefit on the AMD progression later in life (Arons, 2009).

Regardless, active investigation was carried out and numerous complement inhibitors were derived for clinical trials. Among them, POT-4 (Potentia Pharmaceuticals) was developed as a C3 inhibitor for wet AMD. Eculizumab (Soliris, Alexion Pharmaceuticals) was developed as a C5 antibody for the treatment of dry AMD. Since eculizumab is a monoclonal antibody, it has to be administered via intravenous infusion to patients with GA or high risk drusen. ARC-1905 (Ophthotech) is an anti-C5 aptamer administrated via intravitreal injection for

Treatments of Dry AMD 281

acuity. This is very different from the assessment of wet AMD drug actions, as the progression of wet AMD is parallel to the loss of visual acuity. Prolongation of dark adaptation is closely correlated to the severity of AMD (Jackson and Edward, 2008; Owsley et al., 2006; Jackson et al., 2002). Dark adaptation is strongly impacted in AMD long before there is any significant loss of visual acuity (Jackson and Edward, 2008). Thus, measurement of dark adaptation is one of the workable ways to measure the drug efficacy for the treatment of dry AMD. The commercially available prototype dark adaptometer (AdaptRx,

It has been found that rod photoreceptor degeneration precedes cone degeneration in early AMD (Owsley et al., 2001; Curcio et al., 1996; Steinmetz et al., 1993; Chen et al., 2004; Jackson et al., 2004) and rod dysfunction may contribute to the later degeneration of cones because of their inter-dependence (Mohand-Said et al., 2001; 1998; Hicks and Sahel,1999). A ten-item night vision questionnaire (NVQ-10) has been developed by Ying et al (2008). Analysis of NVQ-10 implies that the wet AMD and GA may derive from two different disease physiological processes. Because of the ease of assessment, as compared to dark adaptation measurement with machine, assessing night vision symptoms may be useful in identifying patients with early or intermediate AMD at relatively high risk of progression

Accumulation of the number and size of drusen is another parameter used to measure the progression of dry AMD. Since the change in drusen deposits is very slow and difficult to note subjectively, Matched Flicker (EyelC.com) has been developed to record the changes objectively and precisely. Basically, the precise high-tech use in the space science to record minute changes occurred in the sky at any time period has been applied to measure the changes of drusen occurred in the fundus of the same eye. Basically, two retinal images of the same eye from virtually any source can be loaded into Matched Flicker and the changes can be brought to life and observed as easy-to-detect motion. Since very minute change in drusen accumulation can be detected with the machine precisely and objectively, it allows to shorten the time to detect changes in drusen deposits as compared to inaccurate subjective observation with naked eyes in the past. As a result, clinical study of drug efficacy in

Optical Coherence Tomography (OCT) is an advanced technology that allows researchers to measure the increase in volume and area of drusen over time (Yehoshua et al., 2009; OCT, 2011). Information of detailed theory and selected application is available (OCT, 2011) and Spectral Domain OCT (SD-OCT) is particularly useful for monitoring drusen changes in volume and area which can be related to the progression of dry AMD (Yehoshua et al., 2009). This can be used as a novel clinical trial end point for investigation therapies of dry

RPE are critical cells to maintain healthy function of Bruch's membrane and photocells. The degeneration of RPE can be detected by measurement of the c-wave of ERG (Jiang and Chiou, 2007; Peachey et al., 2002). The suppression of c-wave is proportionally related to the

A useful tool for determining patients' vision related function has been developed (Kokame, 2008) to allow the improved sensitivity for detection of even a slight change in visual activity in the stage of early and intermediate stages of dry AMD. The device is called NEI

slowing down the rate of drusen accumulation can be accomplished easily.

Apeliotus Technologies Inc, Atlanta, GA) is now available.

(Ying et al., 2008).

AMD.

deterioration of RPE in dry AMD.

dry and wet AMD. Another drug, anti-Complement Factor D antibody Fab (FCFD4514S) (Genetech/Roche) is in a phase II trial.

For preclinical R&D, JPE (Jerin: Ophthalmic) is developed with peptidomimetic molecular antagonist against C5a receptor. Interleukin-1 blockers (MacuClear) are also under investigation as eye drops for treatment of dry AMD.

For non-specific immune suppression for the treatment of dry AMD, subcutaneous glatiramer acetate (Copaxane, Teva Pharmaceuticals), intravitreal fluocinolone acetonide (Iluvein implant, Alimera Science) and subcutaneous sirolimus (Rapamycin, MacuSight) are under investigation (Arons, 2009).

#### **4.4 Miscellaneous agents**

Fenretinide (ST-602, Sirion Therapeutics) is an oral compound developed to slow down the progression of GA in AMD. It aims to reduce the accumulation of toxins which are end products of retinol (Vitamin A) related biochemical process. These toxins are accumulated in the form of lipofuscin when the elimination process is reduced such as in dry AMD and Stargardt's disease (Radu et al., 2005; Study of Fenretinide, 2007). Instead of slowing down the toxins productions, MC1101 (MacuClear) aims to facilitate the excretion of toxins by increasing the choroidal circulation (See Section 4.1).

Ciliary neurotrophic factors (CNTF, Neurotech Pharmaceuticals) have been developed to retard the progression of neurodegenerative diseases (A Phase II study, 2007). CNTF has been shown to reduce motor neuron loss in mouse and ciliary ganglion neurons in chick eyes. CNTF is delivered by genetically engineered cells that are housed in a patented delivery system called Encapsulated Cell Technology (ECT). They are surgically implanted through the pars plana into the vitreous and anchored to the sclera. It is designed to bypass the blood retinal barrier to reach the back of the eye (NTC201) (ECT Technology, 2007).

Drugs which can change the construction of extra cellular matrix (ECM) or change the balance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may have an effect on angiogenesis process as well (Berglin et al., 2003). These drugs are called ECM modifiers and are still in the experimental stage of development.

#### **5. Clinical protocols for anti-dry AMD drug studies**

There are at least two major obstacles which hinder the development of drugs for the treatment of dry AMD. First, the long time period that is required to observe progression of the disease (Csaky et al., 2008; AREDSRG, 2001), which discourages researchers as well as investors to get involved. Second, the clinical endpoints to show drug efficacy other than visual acuity are difficult to be determined. There are several promising methods under consideration and if approved by FDA, they can facilitate the drug evaluation and development in the future (FDA, 2008a; 2008b).

Dry AMD is an unique chronic disease whose visual acuity does not change much during the early stage of the progression of the disease. Its change does not parallel to the worsening of visual acuity until the late stage of the disease. Consequently, the efficacy of drug action to treat dry AMD is impossible to be assessed based on the changes in visual

dry and wet AMD. Another drug, anti-Complement Factor D antibody Fab (FCFD4514S)

For preclinical R&D, JPE (Jerin: Ophthalmic) is developed with peptidomimetic molecular antagonist against C5a receptor. Interleukin-1 blockers (MacuClear) are also under

For non-specific immune suppression for the treatment of dry AMD, subcutaneous glatiramer acetate (Copaxane, Teva Pharmaceuticals), intravitreal fluocinolone acetonide (Iluvein implant, Alimera Science) and subcutaneous sirolimus (Rapamycin, MacuSight) are

Fenretinide (ST-602, Sirion Therapeutics) is an oral compound developed to slow down the progression of GA in AMD. It aims to reduce the accumulation of toxins which are end products of retinol (Vitamin A) related biochemical process. These toxins are accumulated in the form of lipofuscin when the elimination process is reduced such as in dry AMD and Stargardt's disease (Radu et al., 2005; Study of Fenretinide, 2007). Instead of slowing down the toxins productions, MC1101 (MacuClear) aims to facilitate the excretion of toxins by

Ciliary neurotrophic factors (CNTF, Neurotech Pharmaceuticals) have been developed to retard the progression of neurodegenerative diseases (A Phase II study, 2007). CNTF has been shown to reduce motor neuron loss in mouse and ciliary ganglion neurons in chick eyes. CNTF is delivered by genetically engineered cells that are housed in a patented delivery system called Encapsulated Cell Technology (ECT). They are surgically implanted through the pars plana into the vitreous and anchored to the sclera. It is designed to bypass the blood retinal barrier to reach the back of the eye (NTC201) (ECT Technology, 2007).

Drugs which can change the construction of extra cellular matrix (ECM) or change the balance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may have an effect on angiogenesis process as well (Berglin et al., 2003). These drugs are called ECM modifiers and are still in the experimental stage of development.

There are at least two major obstacles which hinder the development of drugs for the treatment of dry AMD. First, the long time period that is required to observe progression of the disease (Csaky et al., 2008; AREDSRG, 2001), which discourages researchers as well as investors to get involved. Second, the clinical endpoints to show drug efficacy other than visual acuity are difficult to be determined. There are several promising methods under consideration and if approved by FDA, they can facilitate the drug evaluation and

Dry AMD is an unique chronic disease whose visual acuity does not change much during the early stage of the progression of the disease. Its change does not parallel to the worsening of visual acuity until the late stage of the disease. Consequently, the efficacy of drug action to treat dry AMD is impossible to be assessed based on the changes in visual

(Genetech/Roche) is in a phase II trial.

under investigation (Arons, 2009).

**4.4 Miscellaneous agents** 

investigation as eye drops for treatment of dry AMD.

increasing the choroidal circulation (See Section 4.1).

**5. Clinical protocols for anti-dry AMD drug studies** 

development in the future (FDA, 2008a; 2008b).

acuity. This is very different from the assessment of wet AMD drug actions, as the progression of wet AMD is parallel to the loss of visual acuity. Prolongation of dark adaptation is closely correlated to the severity of AMD (Jackson and Edward, 2008; Owsley et al., 2006; Jackson et al., 2002). Dark adaptation is strongly impacted in AMD long before there is any significant loss of visual acuity (Jackson and Edward, 2008). Thus, measurement of dark adaptation is one of the workable ways to measure the drug efficacy for the treatment of dry AMD. The commercially available prototype dark adaptometer (AdaptRx, Apeliotus Technologies Inc, Atlanta, GA) is now available.

It has been found that rod photoreceptor degeneration precedes cone degeneration in early AMD (Owsley et al., 2001; Curcio et al., 1996; Steinmetz et al., 1993; Chen et al., 2004; Jackson et al., 2004) and rod dysfunction may contribute to the later degeneration of cones because of their inter-dependence (Mohand-Said et al., 2001; 1998; Hicks and Sahel,1999). A ten-item night vision questionnaire (NVQ-10) has been developed by Ying et al (2008). Analysis of NVQ-10 implies that the wet AMD and GA may derive from two different disease physiological processes. Because of the ease of assessment, as compared to dark adaptation measurement with machine, assessing night vision symptoms may be useful in identifying patients with early or intermediate AMD at relatively high risk of progression (Ying et al., 2008).

Accumulation of the number and size of drusen is another parameter used to measure the progression of dry AMD. Since the change in drusen deposits is very slow and difficult to note subjectively, Matched Flicker (EyelC.com) has been developed to record the changes objectively and precisely. Basically, the precise high-tech use in the space science to record minute changes occurred in the sky at any time period has been applied to measure the changes of drusen occurred in the fundus of the same eye. Basically, two retinal images of the same eye from virtually any source can be loaded into Matched Flicker and the changes can be brought to life and observed as easy-to-detect motion. Since very minute change in drusen accumulation can be detected with the machine precisely and objectively, it allows to shorten the time to detect changes in drusen deposits as compared to inaccurate subjective observation with naked eyes in the past. As a result, clinical study of drug efficacy in slowing down the rate of drusen accumulation can be accomplished easily.

Optical Coherence Tomography (OCT) is an advanced technology that allows researchers to measure the increase in volume and area of drusen over time (Yehoshua et al., 2009; OCT, 2011). Information of detailed theory and selected application is available (OCT, 2011) and Spectral Domain OCT (SD-OCT) is particularly useful for monitoring drusen changes in volume and area which can be related to the progression of dry AMD (Yehoshua et al., 2009). This can be used as a novel clinical trial end point for investigation therapies of dry AMD.

RPE are critical cells to maintain healthy function of Bruch's membrane and photocells. The degeneration of RPE can be detected by measurement of the c-wave of ERG (Jiang and Chiou, 2007; Peachey et al., 2002). The suppression of c-wave is proportionally related to the deterioration of RPE in dry AMD.

A useful tool for determining patients' vision related function has been developed (Kokame, 2008) to allow the improved sensitivity for detection of even a slight change in visual activity in the stage of early and intermediate stages of dry AMD. The device is called NEI

Treatments of Dry AMD 283

the disease is closely related to numerous normal physiological functions, such as oxidation, aging, VEGF and PEDF expression, extracellular matrix modifications and the like. Thus, complete suppression of these normal functions is not only unrealistic and even detrimental to induce further devastating side effects. Since these factors are numerous, suppression of one of them can improve the disease only partially to bring it to the borderline, marginal improvement of the disease. The novel idea to solve the problem at the root of the disease is to improve the choroidal circulation which can eliminate all normal metabolic wastes from photocells, Bruch's membrane and RPE cells and to furnish nutrients to these critical tissues. This way, the macular function will go back to normal and the AMD would be reversed or

A phase II study of implants of encapsulated human NTC-201 cells releasing ciliary

AREDSRG (Age-related Eye Disease Study Research Group). A randomized, placebo-

Algvere, P.V., Seregard, S. Age-related maculopathy: pathogenetic features and new

Ambati, J., Ambati, B.K., Yoo, S.H., Ianchulev, S., Adamis, A.P. Age-related macular

AREDS report No. 8. A randomized, placebo-controlled, clinical trial of high-dose

macular degeneration and vision loss. *Arch Ophthalmol*, 2001; 119:1417-1436 Arons, I. AMD update 6: An overview of new treatments for dry AMD.

Berglin, L., Sarman, S., van der Ploeg, I., Steen, B., Ming, Y., Itohara, S., Seregard, S., Kvanta,

Campochiaro, P.A., Nguyen QD, Shah SM, Klein ML, Holz E, Frank RN, Saperstein DA,

treatment modalities. *Acta Ophthalmol Scand*, 2002; 80:136-143

http://www.irvaronsjournal.blogspot.com/2009/12

neurotrophic factor (CNTF) in participants with visual activity impairment associated with atrophic macular degeneration. ClinicalTrials.gov Web site. http://www.clinicaltrials.gov/ct/show/NCT00277134?order=2. Accessed on

controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss. *Archives of* 

degeneration: etiology, pathogenesis, and therapeutic strategies. *Surv Ophthalmol,*

supplementation with vitamins C and E, beta carotene, and zinc for age-related

A. Reduced choroidal neovascular membrane formation in matrix metalloproteinase-2-deficient mice [J]. *Invest Ophthalmol Vis Sci*, 2003; 44:403-408 Bora, P.S., Kaliappan, S., Xu, Q., Kumar, S., Wang, Y., Kaplan, H.J., Bora, N.S. Alcohol linked

to enhanced angiogenesis in rat model of choroidal neovascularization. *Fed Eur* 

Gupta A, Stout JT, Macko J, DiBartolomeo R and Wei LL. Adenoviral vectordelivered pigment epithelium-deprived factor for neovascular age-related macular degeneration: results of phase I clinical trial. *Hum. Gene Ther.* 2006. 17:167-176 CAPT (Complications of Age-Related Macular Degeneration Prevention Trial Research

Group) Laser treatment in patients with bilateral large drusen: the complications of age-related macular degeneration prevention trial. *Ophthalmology.* 2006;113:1974-

suppressed without further development.

October 3, 2007.

2003; 48:257-293

1986.

*Ophthalmology*, 2001; 119:1417-1436

*Biochem Soci J*, 2006; 273: 1403-1414

**7. References** 

VFQ-25 (National Eye Institute Visual Function Questionaire-25) which is responsive to changes in patients' visual activity and is able to differentiate between patients who are responders and those who are not.

Although NEI VFQ-25 measures patients' subjective evaluation of their visual function and how impairment in vision affects their lives, it is reliable, valid and responsive as compared to standard measure of vision used in clinical trials such as BCVA using standardized ETDRS (Early Treatment of diabetic retinopathy study) vision protocols (Kokame, 2008). The NEI VFQ-25 showed a large separation between the groups with improved BCVA (gained >15 letters) stable BCVA (gain or lost 15 < letters) and worse BCVA (lost ≥ 15 letters). It may also provide a more broad assessment of the visual function on life style and vision dependent activities than BCVA alone. On average, the 25-letter or better improvement in BCVA corresponds to an increase in the NEI VFQ-25 score of 8.2 in the MARINA Trial (Minimally Classic/Occult Trial of Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD).

Although not all dry AMD would develop into wet AMD, 10-15% of dry AMD would eventually be converted into wet AMD. Thus, prevention of dry AMD to be converted into wet AMD is also a measurement of drug action to suppress the progression of dry to wet AMD. Since only 10-15% of dry AMD would be converted into wet AMD, a large number of patients are needed for this study to see the difference in drug treatment. In short, ideal clinical endpoints are urgently needed for the measurement of the efficacy of new drugs for the treatment of dry AMD. If approved by FDA, it can shorten the measurement of drug efficacy to save time, effort and funds.

#### **6. Conclusions**

Great deals of efforts have been poured in, in order to elucidate the etiology and pathogenesis of the dreadful disease, age-related macular degeneration, with a hope to develop an effective means of treatment and/or prevention of the disease. Although the etiology and pathogenesis have been largely revealed, its treatment with physical means has since failed and receded to the second line of treatment options. Fortunately, pharmacological agents are now available for the treatment of wet AMD. However, wet AMD is the very late stage of AMD and is too late to save the eyesight for normal daily function. Besides, wet AMD consists of only 10-15% of total AMD patients. Thus, developing efficacious drugs for dry AMD is most urgently needed. The key stage of the treatment of AMD is obviously at the early stage or dry phase of the disease. There are numerous groups of scientists working very hard to develop an efficacious drug yet none have succeeded as of yet.

The major obstacles for the development of ideal drugs for dry AMD are at least two folds. First, AMD is a long term chronic disease with little worsening of visual acuity until the very late stage of the disease. Thus, the clinical end point to measure improvement of visual acuity by drugs at early or even middle stages of the disease is very difficult. The agents for improving the choroidal circulation are under investigation. Alternative end points to measure visual functions have been developed including the measurement of dark adaptation for rod cell functions, the determination of c-wave of ERG for RPE cell functions and macular stress test for cone cell function in the macula. Secondly, the pathogenesis of

VFQ-25 (National Eye Institute Visual Function Questionaire-25) which is responsive to changes in patients' visual activity and is able to differentiate between patients who are

Although NEI VFQ-25 measures patients' subjective evaluation of their visual function and how impairment in vision affects their lives, it is reliable, valid and responsive as compared to standard measure of vision used in clinical trials such as BCVA using standardized ETDRS (Early Treatment of diabetic retinopathy study) vision protocols (Kokame, 2008). The NEI VFQ-25 showed a large separation between the groups with improved BCVA (gained >15 letters) stable BCVA (gain or lost 15 < letters) and worse BCVA (lost ≥ 15 letters). It may also provide a more broad assessment of the visual function on life style and vision dependent activities than BCVA alone. On average, the 25-letter or better improvement in BCVA corresponds to an increase in the NEI VFQ-25 score of 8.2 in the MARINA Trial (Minimally Classic/Occult Trial of Anti-VEGF Antibody Ranibizumab in the

Although not all dry AMD would develop into wet AMD, 10-15% of dry AMD would eventually be converted into wet AMD. Thus, prevention of dry AMD to be converted into wet AMD is also a measurement of drug action to suppress the progression of dry to wet AMD. Since only 10-15% of dry AMD would be converted into wet AMD, a large number of patients are needed for this study to see the difference in drug treatment. In short, ideal clinical endpoints are urgently needed for the measurement of the efficacy of new drugs for the treatment of dry AMD. If approved by FDA, it can shorten the measurement of drug

Great deals of efforts have been poured in, in order to elucidate the etiology and pathogenesis of the dreadful disease, age-related macular degeneration, with a hope to develop an effective means of treatment and/or prevention of the disease. Although the etiology and pathogenesis have been largely revealed, its treatment with physical means has since failed and receded to the second line of treatment options. Fortunately, pharmacological agents are now available for the treatment of wet AMD. However, wet AMD is the very late stage of AMD and is too late to save the eyesight for normal daily function. Besides, wet AMD consists of only 10-15% of total AMD patients. Thus, developing efficacious drugs for dry AMD is most urgently needed. The key stage of the treatment of AMD is obviously at the early stage or dry phase of the disease. There are numerous groups of scientists working very hard to develop an efficacious drug yet none

The major obstacles for the development of ideal drugs for dry AMD are at least two folds. First, AMD is a long term chronic disease with little worsening of visual acuity until the very late stage of the disease. Thus, the clinical end point to measure improvement of visual acuity by drugs at early or even middle stages of the disease is very difficult. The agents for improving the choroidal circulation are under investigation. Alternative end points to measure visual functions have been developed including the measurement of dark adaptation for rod cell functions, the determination of c-wave of ERG for RPE cell functions and macular stress test for cone cell function in the macula. Secondly, the pathogenesis of

responders and those who are not.

Treatment of Neovascular AMD).

efficacy to save time, effort and funds.

**6. Conclusions** 

have succeeded as of yet.

the disease is closely related to numerous normal physiological functions, such as oxidation, aging, VEGF and PEDF expression, extracellular matrix modifications and the like. Thus, complete suppression of these normal functions is not only unrealistic and even detrimental to induce further devastating side effects. Since these factors are numerous, suppression of one of them can improve the disease only partially to bring it to the borderline, marginal improvement of the disease. The novel idea to solve the problem at the root of the disease is to improve the choroidal circulation which can eliminate all normal metabolic wastes from photocells, Bruch's membrane and RPE cells and to furnish nutrients to these critical tissues. This way, the macular function will go back to normal and the AMD would be reversed or suppressed without further development.

#### **7. References**


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**15** 

*Korea* 

**Promising Treatment Strategies** 

*Department of Ophthalmology, Yeouido St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul,* 

Young Gun Park, Hyun Wook Ryu, Seungbum Kang and Young Jung Roh

**for Neovascular AMD: Anti-VEGF Therapy** 

Age-related macular degeneration (AMD) is one of the leading causes of substantial and irreversible vision loss. The prevalence of AMD can be expected to increase along with life expectancy, which has risen steadily [1, 2]. Without treatment, the neovascular form of AMD leads to severe quality-of-life loss within a short time period and considerable economic burden. Vascular endothelial growth factor (VEGF) is a key mediator involved in the control of angiogenesis and vascular permeability and has been shown to be induced by hypoxia in cultured human retinal pigment epithelium (RPE) [3]. VEGF-A is the most potent promoter of angiogenesis and vascular permeability within the VEGF family and its role in the pathogenesis of neovascular AMD is well recognized [4, 5]. The advent of intravitreous VEGF inhibitors has revolutionized the management of neovascular AMD. Yet, frequently, indefinite injections of VEGF blocking agents introduce a significant treatment burden for patients with neovascular AMD, and may potentially put patients in the risk of developing ocular and systemic adverse effects from injections. Many studies on modified treatment regimens have been performed in an attempt to mitigate this burden without compromise to visual acuity outcomes. Meanwhile, various randomized clinical trials on combination

VEGF plays an important role in a variety of in vitro processes, including angiogenesis, microvascular permeability, and endothelial cell survival. On the other hand, these activities are all essential to survival, VEGF has been linked to a number of pathogenic conditions, including

Three VEGF therapies are currently used for the treatment of patients with neovascular AMD: pegaptanib (Macugen, OSI Pharmaceuticals, USA), ranibizumab (Lucentis,

therapies and efforts to develop new pharmacologic agents are ongoing.

**2. Therapeutic monoclonal antibodies and fragments 2.1 Intravitreal ranibizumab and bevacizumab monotherapy** 

Genentech, USA), and bevacizumab (Avastin, Genentech, USA).

**2.1.1 Vascular endothelial growth factor** 

neovascular AMD, diabetic retinopathy, and cancer.

**1. Introduction** 


## **Promising Treatment Strategies for Neovascular AMD: Anti-VEGF Therapy**

Young Gun Park, Hyun Wook Ryu, Seungbum Kang and Young Jung Roh *Department of Ophthalmology, Yeouido St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea* 

### **1. Introduction**

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

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choroidal neovascularization (CNV) in vivo and endothelial cells culture in vitro.

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photooxidative processes initiated in retinal pigment epithelial cells by a lipofuscin

formation of choroidal neovascularization (CNV) in age-related macular

choroidal neovascularization in vivo and endothelial cell culture in vitro. *Current* 

experimental choroidal neovascularization (CNV) in vivo and endothelial cells

Age-related macular degeneration (AMD) is one of the leading causes of substantial and irreversible vision loss. The prevalence of AMD can be expected to increase along with life expectancy, which has risen steadily [1, 2]. Without treatment, the neovascular form of AMD leads to severe quality-of-life loss within a short time period and considerable economic burden.

Vascular endothelial growth factor (VEGF) is a key mediator involved in the control of angiogenesis and vascular permeability and has been shown to be induced by hypoxia in cultured human retinal pigment epithelium (RPE) [3]. VEGF-A is the most potent promoter of angiogenesis and vascular permeability within the VEGF family and its role in the pathogenesis of neovascular AMD is well recognized [4, 5]. The advent of intravitreous VEGF inhibitors has revolutionized the management of neovascular AMD. Yet, frequently, indefinite injections of VEGF blocking agents introduce a significant treatment burden for patients with neovascular AMD, and may potentially put patients in the risk of developing ocular and systemic adverse effects from injections. Many studies on modified treatment regimens have been performed in an attempt to mitigate this burden without compromise to visual acuity outcomes. Meanwhile, various randomized clinical trials on combination therapies and efforts to develop new pharmacologic agents are ongoing.

#### **2. Therapeutic monoclonal antibodies and fragments**

#### **2.1 Intravitreal ranibizumab and bevacizumab monotherapy**

#### **2.1.1 Vascular endothelial growth factor**

VEGF plays an important role in a variety of in vitro processes, including angiogenesis, microvascular permeability, and endothelial cell survival. On the other hand, these activities are all essential to survival, VEGF has been linked to a number of pathogenic conditions, including neovascular AMD, diabetic retinopathy, and cancer.

Three VEGF therapies are currently used for the treatment of patients with neovascular AMD: pegaptanib (Macugen, OSI Pharmaceuticals, USA), ranibizumab (Lucentis, Genentech, USA), and bevacizumab (Avastin, Genentech, USA).

Promising Treatment Strategies for Neovascular AMD: Anti-VEGF Therapy 291

hypertension was low. Over the 24 months trial period, the rates in the ranibizumab 0.5 mg treatment group of the ANCHOR, MARINA, and PIER trials was 5.0 %, 4.6 %, and 0 %,

The pivotal phase III Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA) [12] and the Anti-VEGF Antibody for the Treatment of Predominantly Classic CNV in AMD (ANCHOR) trial [13, 14] demonstrated best-corrected visual acuity (BCVA) outcomes were far superior to any previously published study in the treatment of this disease. At the end of 24 months in the MARINA trial, significantly more ranibizumab-treated patients had maintained [lost <15 Early Treatment Diabetic Retinopathy Study (ETDRS) letters] or improved vision than sham-injected patients. Indeed, 90–95% of patients treated with 0.3 and 0.5 mg ranibizumab maintained vision compared with 53–64% of control patients. Over the same period, vision improved in 25–34% of treated eyes, compared with 4–5% of sham-injected

In the ANCHOR trial, ranibizumab was compared with verteporfin photodynamic therapy (PDT) and demonstrated similar findings: 90–96% of the ranibizumab-treated versus 64–66% of the PDT-treated patients maintained vision, whereas 34–41% versus 6% of each group,

In both trials, a biphasic treatment effect was observed, with the majority of the visual gain achieved in the first 3 months of treatment (the loading phase) followed by stabilization of

Patient-reported outcomes were also assessed in the ANCHOR and MARINA trials to measure the influence of the ranibizumab-mediated improvement in visual acuity (VA) on quality of life. The data demonstrated that patients treated with ranibizumab were more likely to report improvements in near activities, distance activities, and vision specific dependency which were maintained over the 2 year duration of the trial [15, 16]. These data demonstrate that the clinical improvements seen with ranibizumab treatment translate into

More recently, the anatomical benefit of ranibizumab treatment in both the MARINA and ANCHOR studies with regard to angiographic and optical coherence tomography (OCT) characteristics has also been demonstrated. [12, 15, 16] Both functional and anatomical improvements were maintained over the 24 month study period with monthly injections.

Bevacizumab, the predecessor of ranibizumab, is a full-length monoclonal antibody that binds to and blocks the action of all VEGF isoforms. Numerous retrospective [17-20] and prospective studies [21-23] of intravitreal bevacizumab have reported its efficacy for neovascular AMD and low rates of treatment related complications [24]. Although a number of these studies were uncontrolled, relatively small in sample size, of limited follow-up, and varied with regard to outcome measures and retreatment criteria, the reported efficacy of bevacizumab coupled with its low cost when utilized as an intraocular agent has propelled

These outcomes were significantly better than those achieved by the control groups.

respectively, compared with 4.2 %, 3.8 %, and 0 % in the control group.

**2.1.3 Efficacy** 

patients.

respectively, gained more than 15 letters.

the gain (the maintenance phase).

meaningful benefits for the patient.

its adoption worldwide.

Pegaptanib is an oligonucleotide aptamer and was the first VEGF antagonist to be approved by the US Food and Drug Administration (FDA) for use in wet AMD. However, wet AMD patients treated with pegaptanib still experience visual decline. [2, 6] The first monoclonal antibody developed to target VEGF was bevacizumab, a humanized murine monoclonal antibody. Bevacizumab was initially developed for applications in oncology, and received approval as a first-line therapy for widespread colorectal cancer from the US FDA in 2004. Bevacizumab has subsequently been approved for use in non-small cell lung cancer and breast cancer.

The successful development of VEGF as an oncology target led to interest in the potential of anti-VEGF treatment for other therapeutic indications, including ocular neovascular disorders. VEGF-A has been identified as the primary angiogenesis mediator in the eye. It is implicated in ocular neovascularization through its promotion of blood vessel formation and permeability. A role for VEGF-A in neovascular AMD is suggested by immunohistochemistry localization in human choroidal neovascular (CNV) lesions and extrapolation from other disease models [5, 7-9].

New blood vessel formation and leakage play important roles in the development of the neovascular form of AMD, and clinical trials of agents that block VEGF-A activity have produced more evidence that VEGF-A is important in development of this disease.

Ranibizumab is a humanized antibody fragment against VEGF which was specifically designed for intraocular use as a smaller antibody fragment to penetrate through the retina better. The Food and Drug Administration (FDA) approved ranibizumab for treatment of subfoveal neovascular AMD in June, 2006. It was the first drug for AMD treatment shown to improve visual acuity in a substantial percentage of patients.

Bevacizumab is a recombinant humanized monoclonal immunoglobulin antibody that inhibits the activity of VEGF. It has a similar action and is related to the ranibizumab compound with respect to its structure. Bevacizumab was approved by the FDA for the treatment of metastatic colorectal cancer in 2004, but it has not been licensed for the treatment of wet AMD or any other ocular conditions. However, it is recently used off-label worldwide not only for wet AMD but also for other ocular disease entities associated with macular edema and abnormal vessel growth.

Intraocular pharmacokinetic data derived from studies in monkeys demonstrated that through intravitreal administration, ranibizumab distributed rapidly to the retina and had a vitreous half-life of 3 days. Studies in rabbits have demonstrated that ranibizumab can rapidly penetrate through the retina to reach the choroid, just 1 hr after intravitreal admidistration [10]. In primates, serum ranibizumab levels were found to be more than 1000-fold lower than in the vitreous and aquous humor following a single intravitreal injection [11]. These were negligible and tissue concentrations were undetectable.

#### **2.1.2 Safety**

Systemic VEGF inhibition is suspected to be associated with an increased risk of hypertension and arterial thromboembolic events. Given the average age of patients requiring treatment for AMD, it is important that their treatment does not significantly increase the risk of these events. The rate of arterial thromboembolic events and hypertension was low. Over the 24 months trial period, the rates in the ranibizumab 0.5 mg treatment group of the ANCHOR, MARINA, and PIER trials was 5.0 %, 4.6 %, and 0 %, respectively, compared with 4.2 %, 3.8 %, and 0 % in the control group.

#### **2.1.3 Efficacy**

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

Pegaptanib is an oligonucleotide aptamer and was the first VEGF antagonist to be approved by the US Food and Drug Administration (FDA) for use in wet AMD. However, wet AMD patients treated with pegaptanib still experience visual decline. [2, 6] The first monoclonal antibody developed to target VEGF was bevacizumab, a humanized murine monoclonal antibody. Bevacizumab was initially developed for applications in oncology, and received approval as a first-line therapy for widespread colorectal cancer from the US FDA in 2004. Bevacizumab has subsequently been approved for use in non-small cell lung cancer and

The successful development of VEGF as an oncology target led to interest in the potential of anti-VEGF treatment for other therapeutic indications, including ocular neovascular disorders. VEGF-A has been identified as the primary angiogenesis mediator in the eye. It is implicated in ocular neovascularization through its promotion of blood vessel formation and permeability. A role for VEGF-A in neovascular AMD is suggested by immunohistochemistry localization in human choroidal neovascular (CNV) lesions and

New blood vessel formation and leakage play important roles in the development of the neovascular form of AMD, and clinical trials of agents that block VEGF-A activity have

Ranibizumab is a humanized antibody fragment against VEGF which was specifically designed for intraocular use as a smaller antibody fragment to penetrate through the retina better. The Food and Drug Administration (FDA) approved ranibizumab for treatment of subfoveal neovascular AMD in June, 2006. It was the first drug for AMD treatment shown to

Bevacizumab is a recombinant humanized monoclonal immunoglobulin antibody that inhibits the activity of VEGF. It has a similar action and is related to the ranibizumab compound with respect to its structure. Bevacizumab was approved by the FDA for the treatment of metastatic colorectal cancer in 2004, but it has not been licensed for the treatment of wet AMD or any other ocular conditions. However, it is recently used off-label worldwide not only for wet AMD but also for other ocular disease entities associated with

Intraocular pharmacokinetic data derived from studies in monkeys demonstrated that through intravitreal administration, ranibizumab distributed rapidly to the retina and had a vitreous half-life of 3 days. Studies in rabbits have demonstrated that ranibizumab can rapidly penetrate through the retina to reach the choroid, just 1 hr after intravitreal admidistration [10]. In primates, serum ranibizumab levels were found to be more than 1000-fold lower than in the vitreous and aquous humor following a single intravitreal

Systemic VEGF inhibition is suspected to be associated with an increased risk of hypertension and arterial thromboembolic events. Given the average age of patients requiring treatment for AMD, it is important that their treatment does not significantly increase the risk of these events. The rate of arterial thromboembolic events and

injection [11]. These were negligible and tissue concentrations were undetectable.

produced more evidence that VEGF-A is important in development of this disease.

breast cancer.

**2.1.2 Safety** 

extrapolation from other disease models [5, 7-9].

macular edema and abnormal vessel growth.

improve visual acuity in a substantial percentage of patients.

The pivotal phase III Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Neovascular AMD (MARINA) [12] and the Anti-VEGF Antibody for the Treatment of Predominantly Classic CNV in AMD (ANCHOR) trial [13, 14] demonstrated best-corrected visual acuity (BCVA) outcomes were far superior to any previously published study in the treatment of this disease. At the end of 24 months in the MARINA trial, significantly more ranibizumab-treated patients had maintained [lost <15 Early Treatment Diabetic Retinopathy Study (ETDRS) letters] or improved vision than sham-injected patients. Indeed, 90–95% of patients treated with 0.3 and 0.5 mg ranibizumab maintained vision compared with 53–64% of control patients. Over the same period, vision improved in 25–34% of treated eyes, compared with 4–5% of sham-injected patients.

In the ANCHOR trial, ranibizumab was compared with verteporfin photodynamic therapy (PDT) and demonstrated similar findings: 90–96% of the ranibizumab-treated versus 64–66% of the PDT-treated patients maintained vision, whereas 34–41% versus 6% of each group, respectively, gained more than 15 letters.

These outcomes were significantly better than those achieved by the control groups.

In both trials, a biphasic treatment effect was observed, with the majority of the visual gain achieved in the first 3 months of treatment (the loading phase) followed by stabilization of the gain (the maintenance phase).

Patient-reported outcomes were also assessed in the ANCHOR and MARINA trials to measure the influence of the ranibizumab-mediated improvement in visual acuity (VA) on quality of life. The data demonstrated that patients treated with ranibizumab were more likely to report improvements in near activities, distance activities, and vision specific dependency which were maintained over the 2 year duration of the trial [15, 16]. These data demonstrate that the clinical improvements seen with ranibizumab treatment translate into meaningful benefits for the patient.

More recently, the anatomical benefit of ranibizumab treatment in both the MARINA and ANCHOR studies with regard to angiographic and optical coherence tomography (OCT) characteristics has also been demonstrated. [12, 15, 16] Both functional and anatomical improvements were maintained over the 24 month study period with monthly injections.

Bevacizumab, the predecessor of ranibizumab, is a full-length monoclonal antibody that binds to and blocks the action of all VEGF isoforms. Numerous retrospective [17-20] and prospective studies [21-23] of intravitreal bevacizumab have reported its efficacy for neovascular AMD and low rates of treatment related complications [24]. Although a number of these studies were uncontrolled, relatively small in sample size, of limited follow-up, and varied with regard to outcome measures and retreatment criteria, the reported efficacy of bevacizumab coupled with its low cost when utilized as an intraocular agent has propelled its adoption worldwide.

Promising Treatment Strategies for Neovascular AMD: Anti-VEGF Therapy 293

month 3 in the maintenance phase with quarterly dosing, there was a gradual decline in mean BCVA to below the pretreatment baseline (2.2 letters) at 12 months, which remained

More recently, the Efficacy and Safety of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (EXCITE) study directly compared the PIER regimen with a fixed monthly treatment arm (0.3 mg ranibizumab) [31]. Although BCVA outcomes in the two quarterly treatment arms fared better than those in the PIER study at 12 months (2.2 and 3.1 letters gain with 0.3 and 0.5 mg ranibizumab, respectively), neither was as good as monthly dosing (0.9 letters gain). These suboptimal results demonstrate that, on average, quarterly treatment is inferior to monthly

Subsequently to the PIER trial, further investigation of a flexible dosing approach was carried out. The EXCITE trial directly compared a maintenance phase of quarterly injections against the monthly regimen. Consistent with previous observations, an initial gain was made in the first 3 months, after which patients receiving monthly injections contributed to gain VA, whilst those receiving quarterly injections showed a decrease from their 3 months

**Study design MARINA ANCHOR PIER EXCITE** 

**Study duration** 24 months 24 months 24 months 12 months

Monthly Monthly Quarterly

**maintenance phase** 9 9 3 9 for control

The current norm in clinical practice with ranibizumab or bevacizumab is to implement an initiation/induction phase followed by an individualized maintenance phase that is modeled after one of two basic approaches: traditional PRN [32] or 'treat and extend' [33, 34]. Traditional PRN involves both regular follow-up and treatment until the macula is more

Monthly for control Quarterly for study

Monthly for control Quarterly for study

3 for study

**Number of patients** 716 423 184 353

**maintenance phase** Monthly Monthly Quarterly

Table 1. Summary table of many different treatment regimen.

unchanged at 24 months [30].

VA levels. **(Table 1.)** 

**Visit regimen in** 

**Ranibizumab regimen in** 

**maintenance phase** 

**No. of injections in** 

treatment; thus, it has never been adopted in practice.

A recent, large, multicenter, randomized prospective study (Bevacizumab for Neovascular Age-Related Macular Degeneration [ABC] trial) that demonstrated MARINA/ANCHORlike results lends further support for its use in neovascular AMD [25, 26]. On the basis of results from the pivotal phase III clinical trials, ranibizumab dosed monthly represents the gold standard to which all other therapeutics and regimens are to be compared. In clinical practice, many retinal physicians have extrapolated the data and continued using bevacizumab. A formal head-to-head comparison of bevacizumab and ranibizumab is being conducted by the National Eye Institute of the National Institute of Health in the Comparisons of Age-Related Macular Degeneration Treatment Trials (CATTs) [27, 28]. The CATT study design includes four treatment arms: either bevacizumab or ranibizumab on a variable schedule means that monthly follow-up and evaluation of fluid based on OCT, and anti-VEGF injection when CNV becomes active and either bevacizumab or ranibizumab on a fixed monthly schedule for 1 year followed by random assignment to either continued monthly injections or a variable schedule based on the treatment response. The primary outcome measure is mean change in BCVA; secondary outcome measures include number of treatments, anatomical changes in the retina, adverse events, and cost. Preliminary results are reported in 2011 and will provide insight into how ranibizumab and bevacizumab compare with each other within the context of either a fixed monthly or traditional pro re nata (PRN) approach. At 1 year, bevacizumab and ranibizumab had equivalent effects on VA when administered according to the same schedule. Bevacizumab administered monthly was equivalent to ranibizumab administered monthly, with 8.0 and 8.5 letters gained, respectively. Bevacizumab administered as needed was equivalent to ranibizumab as needed, with 5.9 and 6.8 letters gained, respectively. Ranibizumab given as needed with monthly evaluation had effects on vision that were equivalent to those of ranibizumab administered monthly, although the comparison between bevacizumab as needed and monthly bevacizumab was inclusive. Differences in rate of serious adverse events require further study.

#### **3. Modified treatment regimens**

The prospect of indefinitely adhering to the monthly treatment schedules of MARINA and ANCHOR has raised ocular and systemic safety concerns as well as convenience and cost issues for patient and physician alike. The identification of alternative dosing strategies capable of reducing the number of required anti-VEGF injections while still achieving VA outcomes similar to those reached in the pivotal trials has since been a subject of great interest.

The observed biphasic treatment effect raised the possibility that, after the initial 3-months loading phase, maintenance of VA gain may be achieved with less frequent treatments. A PIER trial evaluated ranibizumab administered monthly for 3 months, followed by quarterly injections, and compared this with sham treatment. Under this schedule, ranibizumab did provide a significant VA benefit; a significantly greater number of patients achieved VA stabilization at 24 months compared with patients receiving sham treatment. However, subgroup analysis revealed that VA gains observed during the first 3 months of treatment were only maintained in 40% of patients over the duration of the trial, and for the remaining 60% quarterly dosing was not suitable [29, 30]. Results for both ranibizumab doses in the PIER trial (0.3 and 0.5mg) showed an initial mean improvement in BCVA during the initiation phase with monthly dosing, but after

A recent, large, multicenter, randomized prospective study (Bevacizumab for Neovascular Age-Related Macular Degeneration [ABC] trial) that demonstrated MARINA/ANCHORlike results lends further support for its use in neovascular AMD [25, 26]. On the basis of results from the pivotal phase III clinical trials, ranibizumab dosed monthly represents the gold standard to which all other therapeutics and regimens are to be compared. In clinical practice, many retinal physicians have extrapolated the data and continued using bevacizumab. A formal head-to-head comparison of bevacizumab and ranibizumab is being conducted by the National Eye Institute of the National Institute of Health in the Comparisons of Age-Related Macular Degeneration Treatment Trials (CATTs) [27, 28]. The CATT study design includes four treatment arms: either bevacizumab or ranibizumab on a variable schedule means that monthly follow-up and evaluation of fluid based on OCT, and anti-VEGF injection when CNV becomes active and either bevacizumab or ranibizumab on a fixed monthly schedule for 1 year followed by random assignment to either continued monthly injections or a variable schedule based on the treatment response. The primary outcome measure is mean change in BCVA; secondary outcome measures include number of treatments, anatomical changes in the retina, adverse events, and cost. Preliminary results are reported in 2011 and will provide insight into how ranibizumab and bevacizumab compare with each other within the context of either a fixed monthly or traditional pro re nata (PRN) approach. At 1 year, bevacizumab and ranibizumab had equivalent effects on VA when administered according to the same schedule. Bevacizumab administered monthly was equivalent to ranibizumab administered monthly, with 8.0 and 8.5 letters gained, respectively. Bevacizumab administered as needed was equivalent to ranibizumab as needed, with 5.9 and 6.8 letters gained, respectively. Ranibizumab given as needed with monthly evaluation had effects on vision that were equivalent to those of ranibizumab administered monthly, although the comparison between bevacizumab as needed and monthly bevacizumab was inclusive. Differences in rate of serious adverse events require

The prospect of indefinitely adhering to the monthly treatment schedules of MARINA and ANCHOR has raised ocular and systemic safety concerns as well as convenience and cost issues for patient and physician alike. The identification of alternative dosing strategies capable of reducing the number of required anti-VEGF injections while still achieving VA outcomes

The observed biphasic treatment effect raised the possibility that, after the initial 3-months loading phase, maintenance of VA gain may be achieved with less frequent treatments. A PIER trial evaluated ranibizumab administered monthly for 3 months, followed by quarterly injections, and compared this with sham treatment. Under this schedule, ranibizumab did provide a significant VA benefit; a significantly greater number of patients achieved VA stabilization at 24 months compared with patients receiving sham treatment. However, subgroup analysis revealed that VA gains observed during the first 3 months of treatment were only maintained in 40% of patients over the duration of the trial, and for the remaining 60% quarterly dosing was not suitable [29, 30]. Results for both ranibizumab doses in the PIER trial (0.3 and 0.5mg) showed an initial mean improvement in BCVA during the initiation phase with monthly dosing, but after

similar to those reached in the pivotal trials has since been a subject of great interest.

further study.

**3. Modified treatment regimens** 

month 3 in the maintenance phase with quarterly dosing, there was a gradual decline in mean BCVA to below the pretreatment baseline (2.2 letters) at 12 months, which remained unchanged at 24 months [30].

More recently, the Efficacy and Safety of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (EXCITE) study directly compared the PIER regimen with a fixed monthly treatment arm (0.3 mg ranibizumab) [31]. Although BCVA outcomes in the two quarterly treatment arms fared better than those in the PIER study at 12 months (2.2 and 3.1 letters gain with 0.3 and 0.5 mg ranibizumab, respectively), neither was as good as monthly dosing (0.9 letters gain). These suboptimal results demonstrate that, on average, quarterly treatment is inferior to monthly treatment; thus, it has never been adopted in practice.

Subsequently to the PIER trial, further investigation of a flexible dosing approach was carried out. The EXCITE trial directly compared a maintenance phase of quarterly injections against the monthly regimen. Consistent with previous observations, an initial gain was made in the first 3 months, after which patients receiving monthly injections contributed to gain VA, whilst those receiving quarterly injections showed a decrease from their 3 months VA levels. **(Table 1.)** 


Table 1. Summary table of many different treatment regimen.

The current norm in clinical practice with ranibizumab or bevacizumab is to implement an initiation/induction phase followed by an individualized maintenance phase that is modeled after one of two basic approaches: traditional PRN [32] or 'treat and extend' [33, 34]. Traditional PRN involves both regular follow-up and treatment until the macula is more

Promising Treatment Strategies for Neovascular AMD: Anti-VEGF Therapy 295

patients (71.9%) with no visual loss or improved visual acuity was comparable to the

In addition, Gupta et al. evaluated a treat and extend approach with bevacizumab and found nearly identical results at 12 months following a mean of 7.3 injections in the first year [40]. Although various methods for individualizing maintenance therapy have been

**4. Combination therapy: Photodynamic therapy and antivascular endothelial** 

The development and propagation of CNV membranes involve pro-angiogenic factors, vascular permeability molecules, and inflammatory proteins. Current standard treatment with monthly intravitreal injections of anti-VEGF monotherapy can be limited to the angiogenic component of CNV development and burdensome for both the physician and patient. Patients are subjected to increased risk of adverse effects from monthly treatments that may be lessened with treatment options given with less frequency [43]. While current monotherapy with anti-VEGF agents are effective therapy for CNV, their benefits are short-lived as they are unable to regress the lesions completely. Combination therapy with PDT proven to be effective in CNV regression may have a role not only in the treatment of CNV development but also may provide synergy through blocking

PDT was approved in 2000 by the FDA for the treatment of CNV secondary to AMD. Treatment involves intravenous administration of a light-sensitive dye called verteporfin followed by laser-guided, location-specific activation within the CNV membrane. Activation of the verteporfin molecules incite a phototoxic event within blood vessels, induce endothelial cell damage, platelet aggregation, and eventually lead to thrombosis of vascular channels. Treatment size is limited by the greatest linear diameter of the CNV lesion being

Variable factors within PDT treatment regimens include time of laser application and laser fluence. Standard fluence PDT (sfPDT) was commonly employed in the early studies. The Treatment of AMD with PDT (TAP) study showed stabilization but no improvement in vision with this protocol. In addition, other studies have reported that PDT may inadvertently perturb the normal choriocapillaris bordering a pathologic CNV lesion, resulting in up-regulation and expression of VEGF [46, 47]. This collateral damage may potentially be minimized with reduced-fluence PDT (rfPDT) [46]. rfPDT protocols have gained popularity because of its potential for increased CNV membrane selectivity and propensity to cause less surrounding retinal inflammation. The Verteporfin in Minimally Classic (VIM) trial employed both a standard and reduced fluence PDT protocol and showed stability of vision with either treatment over placebo, but it showed a clear trend toward a better visual outcome with rfPDT. In another comparative study, patients treated with rfPDT tended to have lower rates

While PDT is intended to specifically target CNV vessels, collateral damage to surrounding blood vessels may lead to ischemia of healthy tissue. Following PDT of a CNV membrane, induced ischemia can lead to production of pro-angiogenic factors, especially VEGF. Therefore, combining verteporfin PDT and anti-VEGF therapy may be beneficial compared

of severe visual loss and an overall better visual prognosis [46].

proposed, the optimal non-monthly dosing regimen still remains unclear.

percentages in the monthly injection-based studies.

**growth factor therapy** 

adverse effects.

treated [44, 45].

or less free of exudation, with treatment thereafter during the maintenance phase only in the presence of recurrent exudation. The original prospective studies that evaluated a PRN approach to the maintenance phase were the Prospective Optical Coherence Tomography Imaging of Patients with Neovascular AMD Treated with Intra-Ocular Lucentis (PrONTO) study [35] and the Secondary to Age-Related Macular Degeneration (SAILOR) study [36]. More recently, the Study of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (SUSTAIN) study has contributed additional data [37]. In each of these trials, patients received three consecutive, monthly intravitreal injections of ranibizumab for induction, followed by monthly office visits. Thereafter, a PRN maintenance phase adhered to the following retreatment criteria: loss of at least five ETDRS letters, increase in central macular thickness on OCT of at least 100μm, or new hemorrhage.

Of the three studies, the PrONTO study demonstrated the best VA results. The PrONTO study evaluated an OCT guided, variable-dosing regimen with ranibizumab (0.5 mg) and showed that mean VA improved by 9.3 ETDRS letters at 12 months. Over a 2-year period, mean BCVA outcomes were similar to MARINA and ANCHOR with a mean of 9.9 injections (5.6 in the first year and 4.3 in the second). In comparison, results from the SAILOR study were not as good. In this study, the mean change in BCVA at 12 months from baseline was 0.5 and 1.7 letters in the treatment-naive and previously treated groups, respectively, at the 0.3 mg dose and 2.3 letters in both groups at 0.5 mg. It is worth noting that participants were not monitored as closely in SAILOR as compared with PrONTO, averaging nine visits through 1 year and a mean of 4.9 injections.

The 12-month results from SUSTAIN were slightly better than those from SAILOR (mean BCVA from baseline of 3.6 letters), yet still not as good as the monthly treatment trials. In contrast to SAILOR, participants in the SUSTAIN trial were followed monthly (more like PrONTO) and the mean number of injections over the first year was higher at 5.6.

Other relatively large studies using a traditional PRN approach have recently been published [38-40]. An analysis of these reports highlights an important trend: the best visual acuity results come from the study with the greatest mean number of treatments and closest follow-up, whereas the poorest outcomes were observed in the study with the lowest mean number of treatments and office visits. Unlike traditional PRN, a treat and extend approach initially involves regular and frequent treatment until the macula is dry, followed by a gradual extension of the treatment interval and corresponding follow-up visit. Treatment interval extension continues until there are signs of recurrence, at which point the treatment interval is then reduced.

Kang et al. [41, 42] recently published a retrospective analysis that monthly injections were not given in contrast to the three injections during the initial treatment period in the PIER and PrONTO trials. This study showed that visual acuity improved by 0.078 logMAR units and minimized the number of injections given during 12 months of follow-up (a mean of 4.07 injections were given over the 12 months). The decreased need for retreatment is of great benefit to both patients and clinicians. These results may raise doubts about the need for the three initial loading injections. They reported another study [42], the mean number of injections given in the 12 months period was 4.2 (range, 1-6). Patients were also offered reinjection with ranibizumab on an "as needed" basis. Data showed that the percentage of

or less free of exudation, with treatment thereafter during the maintenance phase only in the presence of recurrent exudation. The original prospective studies that evaluated a PRN approach to the maintenance phase were the Prospective Optical Coherence Tomography Imaging of Patients with Neovascular AMD Treated with Intra-Ocular Lucentis (PrONTO) study [35] and the Secondary to Age-Related Macular Degeneration (SAILOR) study [36]. More recently, the Study of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (SUSTAIN) study has contributed additional data [37]. In each of these trials, patients received three consecutive, monthly intravitreal injections of ranibizumab for induction, followed by monthly office visits. Thereafter, a PRN maintenance phase adhered to the following retreatment criteria: loss of at least five ETDRS letters, increase in central macular thickness on OCT of at least

Of the three studies, the PrONTO study demonstrated the best VA results. The PrONTO study evaluated an OCT guided, variable-dosing regimen with ranibizumab (0.5 mg) and showed that mean VA improved by 9.3 ETDRS letters at 12 months. Over a 2-year period, mean BCVA outcomes were similar to MARINA and ANCHOR with a mean of 9.9 injections (5.6 in the first year and 4.3 in the second). In comparison, results from the SAILOR study were not as good. In this study, the mean change in BCVA at 12 months from baseline was 0.5 and 1.7 letters in the treatment-naive and previously treated groups, respectively, at the 0.3 mg dose and 2.3 letters in both groups at 0.5 mg. It is worth noting that participants were not monitored as closely in SAILOR as compared with PrONTO,

The 12-month results from SUSTAIN were slightly better than those from SAILOR (mean BCVA from baseline of 3.6 letters), yet still not as good as the monthly treatment trials. In contrast to SAILOR, participants in the SUSTAIN trial were followed monthly (more like

Other relatively large studies using a traditional PRN approach have recently been published [38-40]. An analysis of these reports highlights an important trend: the best visual acuity results come from the study with the greatest mean number of treatments and closest follow-up, whereas the poorest outcomes were observed in the study with the lowest mean number of treatments and office visits. Unlike traditional PRN, a treat and extend approach initially involves regular and frequent treatment until the macula is dry, followed by a gradual extension of the treatment interval and corresponding follow-up visit. Treatment interval extension continues until there are signs of recurrence, at which point the treatment

Kang et al. [41, 42] recently published a retrospective analysis that monthly injections were not given in contrast to the three injections during the initial treatment period in the PIER and PrONTO trials. This study showed that visual acuity improved by 0.078 logMAR units and minimized the number of injections given during 12 months of follow-up (a mean of 4.07 injections were given over the 12 months). The decreased need for retreatment is of great benefit to both patients and clinicians. These results may raise doubts about the need for the three initial loading injections. They reported another study [42], the mean number of injections given in the 12 months period was 4.2 (range, 1-6). Patients were also offered reinjection with ranibizumab on an "as needed" basis. Data showed that the percentage of

PrONTO) and the mean number of injections over the first year was higher at 5.6.

averaging nine visits through 1 year and a mean of 4.9 injections.

100μm, or new hemorrhage.

interval is then reduced.

patients (71.9%) with no visual loss or improved visual acuity was comparable to the percentages in the monthly injection-based studies.

In addition, Gupta et al. evaluated a treat and extend approach with bevacizumab and found nearly identical results at 12 months following a mean of 7.3 injections in the first year [40]. Although various methods for individualizing maintenance therapy have been proposed, the optimal non-monthly dosing regimen still remains unclear.

#### **4. Combination therapy: Photodynamic therapy and antivascular endothelial growth factor therapy**

The development and propagation of CNV membranes involve pro-angiogenic factors, vascular permeability molecules, and inflammatory proteins. Current standard treatment with monthly intravitreal injections of anti-VEGF monotherapy can be limited to the angiogenic component of CNV development and burdensome for both the physician and patient. Patients are subjected to increased risk of adverse effects from monthly treatments that may be lessened with treatment options given with less frequency [43]. While current monotherapy with anti-VEGF agents are effective therapy for CNV, their benefits are short-lived as they are unable to regress the lesions completely. Combination therapy with PDT proven to be effective in CNV regression may have a role not only in the treatment of CNV development but also may provide synergy through blocking adverse effects.

PDT was approved in 2000 by the FDA for the treatment of CNV secondary to AMD. Treatment involves intravenous administration of a light-sensitive dye called verteporfin followed by laser-guided, location-specific activation within the CNV membrane. Activation of the verteporfin molecules incite a phototoxic event within blood vessels, induce endothelial cell damage, platelet aggregation, and eventually lead to thrombosis of vascular channels. Treatment size is limited by the greatest linear diameter of the CNV lesion being treated [44, 45].

Variable factors within PDT treatment regimens include time of laser application and laser fluence. Standard fluence PDT (sfPDT) was commonly employed in the early studies. The Treatment of AMD with PDT (TAP) study showed stabilization but no improvement in vision with this protocol. In addition, other studies have reported that PDT may inadvertently perturb the normal choriocapillaris bordering a pathologic CNV lesion, resulting in up-regulation and expression of VEGF [46, 47]. This collateral damage may potentially be minimized with reduced-fluence PDT (rfPDT) [46]. rfPDT protocols have gained popularity because of its potential for increased CNV membrane selectivity and propensity to cause less surrounding retinal inflammation. The Verteporfin in Minimally Classic (VIM) trial employed both a standard and reduced fluence PDT protocol and showed stability of vision with either treatment over placebo, but it showed a clear trend toward a better visual outcome with rfPDT. In another comparative study, patients treated with rfPDT tended to have lower rates of severe visual loss and an overall better visual prognosis [46].

While PDT is intended to specifically target CNV vessels, collateral damage to surrounding blood vessels may lead to ischemia of healthy tissue. Following PDT of a CNV membrane, induced ischemia can lead to production of pro-angiogenic factors, especially VEGF. Therefore, combining verteporfin PDT and anti-VEGF therapy may be beneficial compared

Promising Treatment Strategies for Neovascular AMD: Anti-VEGF Therapy 297

inconsistent visual acuity outcome. In particular, the ability to achieve maintenance or improvement in VA with a more convenient every-other-month injection without need for intervening office visits may potentiate a shift in the current management of neovascular AMD. Continuation of the VIEW studies through the second year will assess the various VEGF Trap-Eye doses administered every 3 months, or more often in the case of worsening disease, as per protocol-defined 'quarterly capped PRN' schedule. Based on phase II data, VEGF Trap-Eye seems to be generally well tolerated with no serious drug-related adverse events. In the 157 patients enrolled in phase II trial, there were two deaths (one from preexisting pulmonary hypertension and one from pancreatic carcinoma) and one arterial thromboembolic events (patient with a history of previous stroke), but no serious systemic

In contrast to current anti-VEGF antibodies, which are rapidly cleared, the VEGF Trap-Eye is relatively degraded more slowly. Due to its high binding affinity and the ability to safely inject high doses into the eye, VEGF Trap-Eye may have longer duration of effect in the eye. Its adoption into clinical practice will depend on efficacy at 4 and 8 week intervals. If effective at 4 and 8 week intervals, VEGF Trap-Eye may offer a competitive price advantage over ranibizumab and the opportunity to significantly reduce treatment burden on patients

Blindness secondary to AMD is common across the world and the pathogenesis of this severe condition is not fully understood. However, the advent of anti-VEGF therapy has revolutionized therapy in the management of neovascular AMD. The appropriate method, dose, regimen, types of combination therapy, and the safety of anti-VEGF remain to be investigated but randomized trials are pending and may provide a clearer answer, which hopefully can help in the treatment of resistant CNV with longer time between treatments.

[1] Bressler, N.M., *Age-related macular degeneration is the leading cause of blindness.* JAMA, 2004.

[2] Emerson, M.V. and A.K. Lauer, *Current and emerging therapies for the treatment of age-*

[3] Shima, D.T., et al., *Hypoxic induction of endothelial cell growth factors in retinal cells:* 

[4] Ferrara, N., H.P. Gerber, and J. LeCouter, *The biology of VEGF and its receptors.* Nat Med,

[5] Kliffen, M., et al., *Increased expression of angiogenic growth factors in age-related maculopathy.*

[6] Gragoudas, E.S., et al., *Pegaptanib for neovascular age-related macular degeneration.* N Engl J

[7] Aiello, L.P., et al., *Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders.* N Engl J Med, 1994. 331(22): p. 1480-7. [8] Boyd, S.R., et al., *Correlation of increased vascular endothelial growth factor with neovascularization and permeability in ischemic central vein occlusion.* Arch Ophthalmol, 2002. 120(12): p. 1644-50.

*identification and characterization of vascular endothelial growth factor (VEGF) as the* 

*related macular degeneration.* Clin Ophthalmol, 2008. 2(2): p. 377-88.

events occurred related to VEGF Trap-Eye [51].

and physicians.

**6. Conclusion** 

**7. References** 

291(15): p. 1900-1.

2003. 9(6): p. 669-76.

*mitogen.* Mol Med, 1995. 1(2): p. 182-93.

Br J Ophthalmol, 1997. 81(2): p. 154-62.

Med, 2004. 351(27): p. 2805-16.

with either modality alone, yielding longer treatment-free intervals and requiring fewer intravitreal injections [44].

The RhuFab V2 Ocular Treatment Combining the Use of Visudyne to Evaluate Safety (FOCUS) study is a multicenter, randomized, single-blind study designed to evaluate the safety and efficacy of sfPDT in combination with intravitreal ranibizumab [48, 49]. It compared sfPDT to combination sfPDT and intravitreal ranibizumab in the treatment of predominantly classic CNV secondary to AMD. One-year data showed greater visual stability in the patients treated with combination therapy and 23.8% of patients experienced improvement in visual acuity, compared with 5% of patients treated with PDT monotherapy alone. The number of retreatments with sfPDT were decreased as well with 91% of patients treated with sfPDT monotherapy requiring repeat treatment while only 28% of patients treated with combination therapy requiring re-treatment within one year. Two-year data showed similar results with 88% of combination treated patients losing less than 15 lines of vision versus 75% of sfPDT alone treated patients. Combination therapy required an average of 0.4 repeat PDT treatments compared with an average of 3.0 in the sfPDT group [49].

#### **5. Vascular endothelial growth factor Trap-Eye**

The most effective dosing regimen and monitoring program for anti-VEGF therapy has yet to be firmly established but new treatments are aimed at extending and improving on the efficacy of ranibizumab. VEGF Trap-Eye (aflibercept, Regeneron Pharmaceuticals, USA) is a promising new anti-VEGF drug. Structurally, VEGF Trap-Eye is a fusion protein of key binding domains of human VEGF receptor 1 and 2 combined with a human IgG Fc fragment. Functionally, VEGF Trap-Eye acts as a receptor decoy with high affinity for all VEGF isoforms, binding more tightly. VEGF Trap-Eye differs from established anti-VEGF therapies in its higher binding affinity for VEGF-A and its blockage of placental growth factors-1 and -2 [50, 51].

Recently, the 1 year results of two parallel randomized, double-masked phase 3 clinical trials (VIEW 1 and VIEW 2) on the efficacy and safety of VEGF Trap-Eye for the treatment of neovascular AMD were reported [51]. Phase I data demonstrated acceptable safety and tolerability of VEGF Trap-Eye in the treatment of neovascular AMD. In Phase II study data, patients dosed in a similar fashion to the PrONTO trial demonstrated stabilization of their vision that was similar to previous studies of ranibizumab at 1 year. All dosing regimens of VEGF Trap-Eye, including 2 mg bimonthly met the primary endpoint of non inferiority compared with monthly 0.5 mg ranibizumab with regard to the percentage of patients with maintenance (loss of <15 ETDRS letters) or improvement in vision. The all treatment groups showed a mean gain of 5.3 letters at 1 year. A greater mean improvement in VA compared with monthly 0.5 mg ranibizumab at 1 year versus baseline represented the secondary endpoint of the study. In both the North American study (VIEW 1) and international study (VIEW 2), more than 95% of patients in each of the following VEGF Trap-Eye dosing groups achieved maintenance of vision compared with 94% of patients on monthly ranibizumab: 0.5 mg monthly, 2 mg monthly, and 2 mg every 2 months. In VIEW 1, patients on 2 mg monthly dosing achieved the secondary endpoint with a mean gain of 10.9 ETDRS letters compared with 8.1 for monthly ranibizumab [51].

The results of the VIEW studies come at a critical time, when clinical evidence suggests that less frequent dosing of existing anti-VEGF therapy, particularly in the first year, may yield inconsistent visual acuity outcome. In particular, the ability to achieve maintenance or improvement in VA with a more convenient every-other-month injection without need for intervening office visits may potentiate a shift in the current management of neovascular AMD. Continuation of the VIEW studies through the second year will assess the various VEGF Trap-Eye doses administered every 3 months, or more often in the case of worsening disease, as per protocol-defined 'quarterly capped PRN' schedule. Based on phase II data, VEGF Trap-Eye seems to be generally well tolerated with no serious drug-related adverse events. In the 157 patients enrolled in phase II trial, there were two deaths (one from preexisting pulmonary hypertension and one from pancreatic carcinoma) and one arterial thromboembolic events (patient with a history of previous stroke), but no serious systemic events occurred related to VEGF Trap-Eye [51].

In contrast to current anti-VEGF antibodies, which are rapidly cleared, the VEGF Trap-Eye is relatively degraded more slowly. Due to its high binding affinity and the ability to safely inject high doses into the eye, VEGF Trap-Eye may have longer duration of effect in the eye. Its adoption into clinical practice will depend on efficacy at 4 and 8 week intervals. If effective at 4 and 8 week intervals, VEGF Trap-Eye may offer a competitive price advantage over ranibizumab and the opportunity to significantly reduce treatment burden on patients and physicians.

#### **6. Conclusion**

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

with either modality alone, yielding longer treatment-free intervals and requiring fewer

The RhuFab V2 Ocular Treatment Combining the Use of Visudyne to Evaluate Safety (FOCUS) study is a multicenter, randomized, single-blind study designed to evaluate the safety and efficacy of sfPDT in combination with intravitreal ranibizumab [48, 49]. It compared sfPDT to combination sfPDT and intravitreal ranibizumab in the treatment of predominantly classic CNV secondary to AMD. One-year data showed greater visual stability in the patients treated with combination therapy and 23.8% of patients experienced improvement in visual acuity, compared with 5% of patients treated with PDT monotherapy alone. The number of retreatments with sfPDT were decreased as well with 91% of patients treated with sfPDT monotherapy requiring repeat treatment while only 28% of patients treated with combination therapy requiring re-treatment within one year. Two-year data showed similar results with 88% of combination treated patients losing less than 15 lines of vision versus 75% of sfPDT alone treated patients. Combination therapy required an average of 0.4 repeat PDT treatments

The most effective dosing regimen and monitoring program for anti-VEGF therapy has yet to be firmly established but new treatments are aimed at extending and improving on the efficacy of ranibizumab. VEGF Trap-Eye (aflibercept, Regeneron Pharmaceuticals, USA) is a promising new anti-VEGF drug. Structurally, VEGF Trap-Eye is a fusion protein of key binding domains of human VEGF receptor 1 and 2 combined with a human IgG Fc fragment. Functionally, VEGF Trap-Eye acts as a receptor decoy with high affinity for all VEGF isoforms, binding more tightly. VEGF Trap-Eye differs from established anti-VEGF therapies in its higher binding affinity for VEGF-A and its blockage of placental growth

Recently, the 1 year results of two parallel randomized, double-masked phase 3 clinical trials (VIEW 1 and VIEW 2) on the efficacy and safety of VEGF Trap-Eye for the treatment of neovascular AMD were reported [51]. Phase I data demonstrated acceptable safety and tolerability of VEGF Trap-Eye in the treatment of neovascular AMD. In Phase II study data, patients dosed in a similar fashion to the PrONTO trial demonstrated stabilization of their vision that was similar to previous studies of ranibizumab at 1 year. All dosing regimens of VEGF Trap-Eye, including 2 mg bimonthly met the primary endpoint of non inferiority compared with monthly 0.5 mg ranibizumab with regard to the percentage of patients with maintenance (loss of <15 ETDRS letters) or improvement in vision. The all treatment groups showed a mean gain of 5.3 letters at 1 year. A greater mean improvement in VA compared with monthly 0.5 mg ranibizumab at 1 year versus baseline represented the secondary endpoint of the study. In both the North American study (VIEW 1) and international study (VIEW 2), more than 95% of patients in each of the following VEGF Trap-Eye dosing groups achieved maintenance of vision compared with 94% of patients on monthly ranibizumab: 0.5 mg monthly, 2 mg monthly, and 2 mg every 2 months. In VIEW 1, patients on 2 mg monthly dosing achieved the secondary endpoint with a mean gain of 10.9 ETDRS letters

The results of the VIEW studies come at a critical time, when clinical evidence suggests that less frequent dosing of existing anti-VEGF therapy, particularly in the first year, may yield

intravitreal injections [44].

factors-1 and -2 [50, 51].

compared with an average of 3.0 in the sfPDT group [49].

**5. Vascular endothelial growth factor Trap-Eye** 

compared with 8.1 for monthly ranibizumab [51].

Blindness secondary to AMD is common across the world and the pathogenesis of this severe condition is not fully understood. However, the advent of anti-VEGF therapy has revolutionized therapy in the management of neovascular AMD. The appropriate method, dose, regimen, types of combination therapy, and the safety of anti-VEGF remain to be investigated but randomized trials are pending and may provide a clearer answer, which hopefully can help in the treatment of resistant CNV with longer time between treatments.

#### **7. References**


Promising Treatment Strategies for Neovascular AMD: Anti-VEGF Therapy 299

[30] Abraham, P., H. Yue, and L. Wilson, *Randomized, double-masked, sham-controlled trial of* 

[31] Schmidt-Erfurth, U., et al., *Efficacy and safety of monthly versus quarterly ranibizumab* 

[32] Spaide, R., *Ranibizumab according to need: a treatment for age-related macular degeneration.*

[33] Brown, D.M. and C.D. Regillo, *Anti-VEGF agents in the treatment of neovascular age-related* 

[34] Chiang, A. and C.D. Regillo, *Preferred therapies for neovascular age-related macular* 

[35] Lalwani, G.A., et al., *A variable-dosing regimen with intravitreal ranibizumab for neovascular* 

[36] Boyer, D.S., et al., *A Phase IIIb study to evaluate the safety of ranibizumab in subjects with neovascular age-related macular degeneration.* Ophthalmology, 2009. 116(9): p. 1731-9. [37] Tano, Y. and M. Ohji, *EXTEND-I: safety and efficacy of ranibizumab in Japanese patients with* 

[38] Dadgostar, H., et al., *Evaluation of injection frequency and visual acuity outcomes for* 

[39] Rothenbuehler, S.P., et al., *Effects of ranibizumab in patients with subfoveal choroidal* 

[40] Gupta, O.P., et al., *A treat and extend regimen using ranibizumab for neovascular age-related* 

[41] Kang, S. and Y.J. Roh, *Ranibizumab treatment administered as needed for occult and* 

[42] Kang, S. and Y.J. Roh, *One-year results of intravitreal ranibizumab for neovascular age-related* 

[43] Beer, P.M., et al., *Intraocular concentration and pharmacokinetics of triamcinolone acetonide after a single intravitreal injection.* Ophthalmology, 2003. 110(4): p. 681-6. [44] Cruess, A.F., et al., *Photodynamic therapy with verteporfin in age-related macular* 

[46] Azab, M., et al., *Verteporfin therapy of subfoveal minimally classic choroidal* 

*pharmacoeconomic properties.* Acta Ophthalmol, 2009. 87(2): p. 118-32. [45] *Verteporfin therapy of subfoveal choroidal neovascularization in age-related macular* 

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*clinical trial.* Arch Ophthalmol, 2005. 123(4): p. 448-57.

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### *Edited by Gui-Shuang Ying*

Age-related Macular Degeneration (AMD) is the leading cause of vision loss and blindness in the developed countries. In the past decade, great progress has been made in understanding the pathobiology and genetics of this blinding disease, as well as in finding new therapies for its treatment. These include the discovery of several genes that are associated with the risk of AMD, new anti-VEGF treatments for wet AMD and new imaging techniques to diagnose and monitor the AMD. All chapters in this book were contributed by outstanding research scientists and clinicians in the area of AMD. I hope this timely book will provide the basic scientists and clinicians with an opportunity to learn about the recent advances in the field of AMD.

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

Age Related

Macular Degeneration

The Recent Advances in Basic Research

and Clinical Care

*Edited by Gui-Shuang Ying*

Photo by Rollopda / iStock