Controversial aspects of photodynamic therapy

Introduction

Photodynamic therapy (PDT) is generating intense interest in ophthalmology with the recent introduction of verteporfin (Visudyne; Novartis AG, Bulach, Switzerland) as a treatment for age-related macular degeneration (AMD).1-3 The two-year results of the Treatment of Age-related Macular Degeneration Study indicated that verteporfin therapy is more likely than placebo to result in stabilization of visual acuity in patients with predominantly classic choroidal neovascular membranes secondary to AMD at two years.4 However, several controversial points in PDT require further and detailed discussion. In this chapter, the following four aspects are discussed: selectivity of photosensitizers for targeting choroidal neovascularization (CNV); pitfalls in the application of PDT in the clinical setting; photosensitizer properties; and future trends in photodynamic and antiangiogenic therapy.

Photosensitizer selectivity: observations based on histological findings

PDT is predicated upon the selective damage of pathological tissue while preserving surrounding normal tissue, and was originally designed to treat solid tumors and their associated neovascular processes.5 PDT consists of local activation of a photosensitizer using laser irradiation of an appropriate wavelength, after that photosensitizer has selectively accumulated in neovascular tissues. This results in activation of singlet and reactive oxygen species and other free radicals that can directly induce vascular cell death or occlude the vascular supply through the activation of the clotting cascade.6-8 The main

pathway of photodynamic damage to cells has been identified as a type II photosensitization reaction: singlet oxygen directly damages the plasma membrane and intracellular membranes (Fig. 1).6,9 Typical values of the decay lifetime of singlet oxygen are 3 µsec in H2O, 30 µsec in deuterium oxide, 12 µsec in ethanol, and 0.2 µsec in living cells.6,9 Therefore, a singlet oxygen molecule can diffuse only about 0.1 µm during its lifetime in tissue, limiting the primary reactions to the initial localization sites.6,9 This is the basis of the selective targeting of PDT within the targeted tissues.

Although sensitizers accumulate in neovascular tissue such as CNV membranes, adjacent tissues, especially retinal pigment epithelium (RPE) cells and photoreceptor cells, are also moderately damaged. This finding was demonstrated by a series of experiments using verteporfin and other photosensitizers (Fig. 2).

Pitfalls of PDT: clinical application

PDT is a straightforward treatment. Patients are administered the dye, either by intravenous infusion (hydrophobic photosensitizers, i.e., verteporfin) or intravenous bolus (hydrophilic photosensitizers, i.e., mono-L-aspartyl chlorin e6 (NPe6)), and then seated at the slit lamp, which is coupled to the appropriate diode laser system. A contact lens is placed on the ocular surface with a coupling agent such as Goniosol or Artificial Tears. At the optimal time, the laser is activated for the treatment duration. Care is taken to use the minimum slit-lamp illumination necessary to identify macular landmarks, thereby minimizing the possibility of inadvertent activation of the photosensitizer by the white light.

There are several factors that can influence the outcome of PDT: intraocular pressure, region of fundus treated, ocular pigmentation, duration of laser exposure, and retreatment.10 Too much pressure when placing the contact lens against the cornea can result in the effect of PDT being diminished.10 Similarly, treatment spots of similar fluence but longer exposure time result in greater angiographic and funduscopic lesions than shorter exposure time and higher power spots.10 Fundus pigmentation has been demonstrated to affect the appearance of treatment spots in pigmented rabbits.10 Additionally, a treatment spot location in the posterior segment is more likely to result in a therapeutic effect than a peripheral lesion.10 Fortunately, unlike in laser photocoagulation, the laser spot does not have to be focused perfectly upon the retina in order to achieve a therapeutic effect.10

If all these factors have been controlled and there does not appear to be an angiographically visible treatment spot within 24 hours, then the possibility of inadvertent subcutaneous injection must be considered. This complication is difficult to miss at the time of treatment, as the clinical signs of infiltration are usually visible in the antecubital region, and can be associated with discomfort at the site.

When retreating patients, it is wise to consider that the treatment may result in cumulative damage to the RPE, photoreceptors, and inner retina.10,11

Selection of photosensitizers: hydrophilic versus hydrophobic sensitizers, mechanisms of dye accumulation, and indocyanine green-guided PDT

Photosensitizers were originally thought to be selective to neoplastic tissue because they were noted to accumulate in solid tumors.12,13 Eventually it was discovered that the tumor vasculature was the primary target of PDT.14,15 This property of biodistribution and treatment selectivity may play a beneficial role in treating subfoveal CNV. However, the firstgeneration photosensitizers were limited by two main factors: suboptimal tissue penetration of the maximum exciting wavelength and prolonged cutaneous phototoxicity.16-19

An advantageous sensitizer should have low skin photosensitization and high photosensitizing ability in targeted tissue when exposed to far-red light. Many second-generation sensitizers have been designed to overcome these problems for clinical application. These second-generation sensitizers possess major absorption peaks at wavelengths above 650 nm.20

Verteporfin, a synthetic chlorin-like porphyrin with a light absorption peak at 692 nm, is a hydrophobic sensitizer in a liposomal preparation for administration by intravenous infusion over ten minutes.1,2 The mechanism of verteporfin uptake by neovascular tissue is not known, but it has been suggested that neovascular and neoplastic tissues have increased lipoprotein receptors that may enhance the preferential uptake of verteporfin.21,22 In the pilot examination using experimental CNV, verteporfin was coupled with low-density lipopro-

tein (LDL) in order to enhance its delivery to neovascular and tumor tissues and its PDT effect.23 CNV may selectively accumulate lipoprotein-asso- ciated sensitizers because of increased LDL receptors in rapidly proliferating endothelium and increased LDL transport across the endothelium of permeable vessels.24,25 Liposomal verteporfin was used in the second series of the experiments and clinical trials which demonstrated the relative selectivity in CNV occlusion.1-4,26,27 This indirect evidence suggests that the verteporfin binds to LDL in the blood and then

accumulates preferentially in neovascular tissue in the choroid.28

Other second-generation sensitizers have been proposed for PDT, and the list continues to grow.29 Representative second-generation sensitizers include phthalocyanine dyes,30,31 purpurins,32 ATX-S10,33 and NPe6, which have all been investigated for the treatment of ocular diseases. Among these dyes, there are significant differences in the time intervals required for peak uptake and retention levels. In a mouse study, peak circulating levels of photosensi-

tizer were reached for hematoporphyrin derivative after five to ten hours, for phthalocyanine dyes after 24-48 hours, for verteporfin after three hours, and for NPe6 after two to 60 minutes.29 Levels of singlet oxygen quantum yields were found to be: hematoporphyrin derivative, 0.29; purpurins, 0.67; phthalocyanine dyes, 0.36; and NPe6, 0.77.29

NPe6 is a second-generation photosensitizer that has undergone preliminary clinical trials in humans.34 The advantages of NPe6 are minimal skin photosensitization, a major absorption peak at a far-red

wavelength, hydrophilic formulation allowing intravenous bolus administration with rapid tissue uptake and clearance, high affinity for neovascular tissues, and a high photosensitizing ability.

We have previously demonstrated that combining NPe6 with a laser at 664 nm allowed efficient occlusion of choroidal vessels with minimal injury to the overlying sensory retina of normal rabbits and monkeys, and that the primary targeted organelle may be the lysosome (Fig. 3).38 This finding agrees with the reports by Roberts and colleagues that NPe6

Fig. 4. Fluorescence microscopy of NPe6. NPe6 accumulated intensively in the choroidal neovascular lesions (arrowhead) and retinal pigment epithelial cells. Some choroidal vascular walls also fluoresced moderately. In contrast, there was trace fluorescence in retinal vascular walls (arrow) and microcapillaries. There was little fluorescence of NPe6 detected in retinal stroma, vascular lumen of the retina and choroid, vitreous cavity and subretinal space. (Reproduced from Mori et al.42 by courtesy of the publisher.)

enters the cell by endocytosis and is localized in lysosomes, whereas other hydrophobic sensitizers enter the cell by diffusion and are located throughout the cytoplasm. These properties on cellular uptake and targeting selectivity might result from the hydrophilic nature of NPe6.39-41 Fluorescence microscopy demonstrated the strong accumulation of NPe6 in CNV and RPE cells,42 possibly indicating active endocytosis in these locations. In contrast, there was trace fluorescence in the retinal vascular walls and the subretinal space, and no fluorescence in the retinal stroma or vitreous cavity (Fig. 4).42 These fluorescence microscopic findings support the hypothesis of the beneficial properties of active accumulation in CNV mediated by the lysosomal uptake, indicating targeting selectivity by NPe6. Biodistribution of NPe6 in the fundus with experimental CNV has also been examined with fundus NPe6 videoangiography, utilizing a scanning laser ophthalmoscope and a 488nm argon laser light that fit the minor absorption peak of NPe6.39 The peak time of dye accumulation in experimental CNV with this system was 20-60 minutes after the dye injection, as evidenced by

increasing fluorescence intensity of retinal vessels after 20-60 minutes, followed by a decline in intensity (Fig. 5a-c). Although the dye concentration used for NPe6 angiography was higher than that in the PDT experiment, these angiographical findings may provide a rough estimation for the optimal timing of the laser irradiation.

An additional finding during the NPe6 angiography experiments was that there was a great similarity in dye-filling, dye accumulation, and retention patterns of NPe6 and indocyanine green (ICG) (Fig. 5a-f). Both NPe6 and ICG have the same biochemical properties: they are hydrophilic, of approximately equivalent molecular weights, and have a high affinity for lipoproteins.38-43 These characteristics may play an important role in the biodistribution of dye, and provide an explanation for the similarity of those angiograms. ICG angiography, which has been applied in a wide spectrum of choroidal diseases (especially AMD), has been proven safe for clinical use,44 and provides an estimation or a simulation of the NPe6 fundus biodistribution in various patients with AMD. This indirect evidence suggests the enhancement of practicality of NPe6 PDT by the guidance of ICG, especially in the determination of optimal timing for laser irradiation. ICG-guided PDT also has advantages (in the detailed parameter settings pointed out in the section on pitfalls in PDT). We may be able to estimate the parameters based on ICG-angiography findings of dye accumulation, fundus pigmentation, and other fundus conditions such as retinal edema, subretinal hemorrhage, and exudates.

Future trends in PDT: prevention of CNV recurrence; modulation of CNV with antiangiogenic therapy

One of the most important problems with current PDT is the high rate of recurrence of CNV.3,4 In the clinical study of a single treatment of verteporfin PDT, fluorescein leakage reappeared in at least a portion of the CNV one to three months after treatment.3 Increasing the photosensitizer concentration or the light doses did not prevent the recurrence and could lead to undesirable, nonselective damage to the retinal vessels.1 The two-year results of large clinical trials showed decreased rates of moderate vision loss; however, 5.6 treatments were needed during the two-year follow-up period.4 The necessity for multiple PDT sessions can be expected to lead to cumulative damage to the RPE and choriocapillaris, and to possible progressive treatmentrelated vision loss.11,45 PDT per se may result in occlusion of CNV, but is not thought to regulate molecular signals (angiogenic stimuli) that mediate the neovascular process; this failing may be the reason for the common recurrence of CNV.

Several antiangiogenic agents are now under investigation for another alternative or additional

modality for subfoveal CNV. Interferon alpha 2a causes dramatic involution of hemangiomas and inhibits iris neovascularization in a model of ischemic retinopathy.46,47 However, a multicenter, randomized, placebo-controlled trial demonstrated that patients with CNV who received interferon alpha 2a did not have any involution of CNV and, at the close of the study, had worse vision than those treated with a placebo.48

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen with a central role in ocular angiogenesis.49-51 Inhibition of VEGF, using antiVEGF antibodies or soluble receptors, can prevent the development of experimental iris or retinal neovascularization.52,53 VEGF kinase inhibitors, which block VEGF signalling, prevent the development of retinal neovascularization and CNV.54 These facts suggest that anti-VEGF therapy may play a role in the prevention of CNV or its recurrence after PDT. Phase I clinical trials testing the safety and tolerability of intraocular injections of an aptamer that binds VEGF or an anti-VEGF antibody have been completed, and phase II trials are being planned.

Purported endogenous inhibitors of angiogenesis have also been described, including angiostatin,55 endostatin,56 antithrombin III,57 pigment epitheliumderived factor (PEDF),58 etc. However, their role, if any, in the development of retinal neovascularization and CNV is unknown. Recently, Mori et al. used adenoviral vectors to demonstrate that two of these proteins, endostatin59 and PEDF,60 inhibit ocular neovascularization. These studies also provided proof of the concept for the use of gene transfer to treat ocular neovascularization. Gene transfer offers a means for local delivery of a therapeutic agent without systemic side-effects or repeated intraocular injections (because of the sustained release of the protein).

Mori et al. have also shown that, in two ocular neovascularization models, transgenic mice expressing VEGF in photoreceptors and a laser-induced CNV model, PEDF gene transfer in eyes with already established neovascularization caused regression of the neovascularization.61 This is the first demonstration of a pharmacological treatment that causes regression of ocular neovascularization, and could potentially be applied to the many patients who present with subfoveal CNV. However, adenoviral vectors have features that may limit their use in humans, including some evidence of toxicity and decreasing transgene expression to low levels over the course of a few months. Patients with AMD are at risk for the development or recurrence of CNV for many years, and long-term treatment is needed. Prolonged intraocular expression has been achieved with adeno-associated viral vectors. Actually, it has also reported that adeno-associated viral vector mediated gene transfer of PEDF inhibits CNV development.62 Phase I clinical trials of gene therapy expressing PEDF are now being planned.63 It is now expected that several drugs or viral vectors for anti-

angiogenic therapy will be able to overcome the problems of PDT and will present an additional or alternative modality for AMD patients.

Conclusions

Photodynamic therapy offers new hope for patients with AMD and other diseases associated with CNV. The primary mechanism of action is selective occlusion of neovascular vessels following activation with a low-energy laser of the appropriate wavelength. While verteporfin is the only drug approved for treatment in humans, photosensitizers such as ATX-10 and NPe6 have shown great promise. Future trends include the use of selective antiangiogenic agents and gene therapy to control CNV, possibly in conjunction with PDT.

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