Photodynamic therapy: basic principles and mechanisms

Abstract

In this article, the authors describe the principles and mechanisms of photodynamic therapy (PDT). After an introduction on the PDT of cancer, the photophysics of photosensitizer molecules is described, followed by the elementary photochemical mechanisms and some of the PDT-induced biomolecular cascades. The authors then focus on the effects of PDT on the blood vessels and the treatment of choroidal neovascularization (CNV) of the retina associated with the exudative form of age-related macular degeneration (AMD). The selectivity of the PDT of CNV in AMD is discussed, and some possible improvements are proposed.

Introduction

The history of photodynamic therapy (PDT) has recently been reviewed,1,2 and excellent overviews on the subject of PDT exist.3-5 PDT for cancer is presented by the schematic description shown in Figure 1, in which the human body is simplified as

athree-compartment system.

Figure 1 shows the PDT of early-stage superfi-

cial non-metastasized cancer in, for example, a hollow organ (lung or esophagus). After (1) injection of a photosensitizer (PS), (2) a certain amount of selective uptake and/or removal leads to the local preferential concentration of the PS in the tumor compared to the surrounding normal tissue. At the

same time, a high concentration of PS may be found in the liver, kidneys, or other organs, by which PS is removed from the body. We call this situation partial selectivity. It is important that PS is applied at concentrations at which, by itself, it is not toxic to any part of the body. PS is then activated (3) by applying light to the surface of the hollow organ. Generally, this is performed in such a way that it shields the superficial normal tissue surrounding the tumor. Light delivery systems for this purpose are described in detail elsewhere.6 The photosensitizer activated by light (PS*) then destroys the tissue in which it is located. (4) The volume of destroyed (mostly necrotic) tissue is largely determined by the area where the light is applied, by the limited penetration of light of a given wavelength into the tissue, and by the selectivity of PS concentration in the tumor compared to the surrounding normal tissues. Thus, the deeper lying tissue below the cancer should not be destroyed and, most importantly, the high concentration of PS in other organs, such as the liver, spleen, or kidneys, is of no consequence, since they are not reached by light. Exceptions to this are, of course, the skin and the eyes, which must be protected by the patient staying out of sunlight

(or strong artificial light) until most PS has been removed from these organs by natural pharmacokinetic processes. Finally, in time, the damaged surface will heal. It should also be underlined that, in themselves, the low light intensities used in PDT are not harmful to the body either.

Very promising results have been obtained using PDT on early stage cancer.7,8 These demonstrate the efficacy and simplicity of this minimally-invasive and repeatable procedure. Furthermore, side-effects, such as skin photosensitivity, stenosis, and perforation of the hollow organs can essentially be completely avoided by using the newest PSs, light applicators, and PDT procedures. In particular, most new PSs are designed to be rapidly removed from the body. Figure 2 shows the ‘selective’ necrosis of an early squamous cell cancer in the bronchi about ten days after PDT.

Tables 1 and 2 demonstrate the low rates of recurrence in this procedure after several years of follow-up using two different PSs for early squamous cell carcinomas (SCC) in the upper aerodigestive tract (UAT), the esophagus, and the bronchi.

The data shown in Tables 1 and 2 show that, while ‘carcinoma in situ’ can essentially be treated with

100% efficacy, recurrence increases as the staging of the cancer advances. This is probably in part due to an increase in distant metastatic disease, which cannot be treated easily due to the limited penetration of light into the tissue. In PDT, the maximum depths of necrosis that have been demonstrated range from several millimeters to about 1 cm, depending on the PS and the excitation wavelength; beyond that, PDT can still be quite effective using interstitial illumination.

Photophysics

Absorption of light by PS or other molecules is governed by the so-called Beer-Lambert law

where I0 is the light intensity at a given wavelength (λ) incident on a sample of thickness l in units of cm, which contains photosensitizer molecules at

rmolesi ε

concentration cu u. is the decadic molar ex- q cm3t

tinction coefficient, which gives the probability that light is absorbed by the PS at wavelength λ. The

cm2 . I is the mole

light intensity transmitted through the sample. γ which is often unity, is a factor that corrects for the nonlinearity of the system, which can be due, for instance, to the width of the exciting light being much broader than the spectral features of the PS.

When a photon is absorbed by a PS it gains energy according to

E = hν = hλc

where E is the photon energy (erg), h is Planck’s constant (erg.sec), ν is the frequency of the light (sec-1), c here is the velocity of light (cm sec-1) and λ is the wavelength in cm.

If we measure the light absorption as a function of the wavelength for a given PS, the resulting curve of ε as a function of wavelength is called an absorption spectrum. This is shown in Figure 3 or a ‘typical’ porphyrin (protoporphyrin IX in this case), together with its chemical structure.

The absorption of a photon by a PS means that the PS energy is increased by the energy of the photon E = hν. In other words, the spectrum in Figure 3 describes the relative probability of photon-induced energy level changes in the PS. The absorption in Figure 3 near 400 nm is called the Soret band, and the other four bands between 500 and 630 nm are called the Q bands. The probability of absorption of a photon by a PS depends upon several properties of the ground state and the state excited by the photon: i.e., on the electronic spin multiplicity, the overlap of the orbitals in space, the symmetry of the wave functions, and the magnitude of change of momentum. The electrons in a molecule are generally found in pairs in spatial volumes called orbitals

1

with either spin (s) equal to + 2 (↑) or spin equal

to – 12 (↓), and the multiplicity of an electronic con-

figuration is defined as 2S + 1 where S = Σ s. In a PS with an even total number of electrons, we have singlet states where 2S + 1 = 2 U$12% + $–21%Y + 1 = 1.

These states may be indicated by (↑↓). We also have triplet states where 2S + 1 = 2 U$ 12 % + $ 12%Y + 1 = 3

which are labelled by (↑↑). Thus, the ground state and the lower electronic states in a PS may typically have the configuration shown in Figure 4.

Photon absorption from the ground singlet state S0 to the first excited singlet state S1 is a process with no change in multiplicity. This is called a spinallowed transition. Following this photon-induced transition to S1 different energy redistribution pathways may occur, as shown schematically in Figure 5, a so-called Jablonski diagram.

The different electronic state are indicated as S0, S1, S2 or T1, and all have sublevels of energy rep-

resenting some excess rotational and vibrational energy. Following absorption of a photon on an extremely short time scale, about 10-15 seconds, the PS is in a vibrationally excited state of S1. This state then undergoes fast (~10-12 seconds) vibrational relaxation (VR) to give the S1 de–excited singlet state. From this ‘ground’ S1 state five different processes can basically occur, depending on the PS:

1.Fluorescence, generally on a time scale of 10-8 to 10-9 seconds. Emission of a fluorescent photon is to one of several rovibrational states of the

S0 ground electronic state. Averaged over many emitted photons, this gives a broad-banded emission, which is somewhat red shifted from the absorption wavelength. This is the so-called Stokes shift.

2.Internal conversion (IC). This is a non-radiative change between states of the same multiplicity.

In Figure 5, the IC process between S1 and vibrationally excited S0 is followed by the loss of vibrational excess energy colliding with the molecules surrounding the PS. Thus, the photon energy is rapidly degraded into heat.

3.Intersystem crossing (ISC). This is also a nonradiative change between electronic states, however, of a different multiplicity. The ISC process indicated in Figure 5 is a non-radiative transfer

between S1 and T1. Due to the energy difference (so-called singlet-triplet splitting), T1 is produced in a vibrationally excited state which once again undergoes rapid VR to give the metastable trip-

let vibrational ‘ground’ state T1. This state can then either undergo a chemical reaction, transfer its energy to another molecule, or internally con-

vert to S0, or radiate in a non-allowed spin-for- bidden transition to one of several rovibrational

states of the S0 ground electronic state. The latter, since it is a spin-forbidden process, is quite slow and is called phosphorescence.

4.S1 can react chemically in many different ways: for instance, it can rearrange itself, it can add itself to another molecule, or dissociate itself.

5.Energy exchange with a neighboring molecule, which is then excited to its singlet, excited state.

Different PS are selected or synthesized for their

different properties, depending on the application. For instance, fluorescein and indocyanine green (ICG) are used for fluorescence angiography in ophthalmology. They are chosen in particular for having a high fluorescence quantum yield. Most PSs used in PDT are chosen for their high yield of long-lived triplet states. The oxygen present in the tissue can diffuse to the triplet (T1) state and pick up its energy by colliding with it. The excited oxygen can then diffuse away from the PS and oxidize certain types of the surrounding molecules (or get quenched). In a given PS, the preferential pathway can be modified by chemical changes being made. For instance, substituting hydrogen atoms by heavy halogen atoms or oxygen by sulphur in the PS macrocycle gives higher triplet yields. This is the case, for instance, when going from fluorescein (a predominantly fluorescing molecule) to tetrachlorotetraiodofluorescein or Bengal rose, which is a good photosensitizer with a high triplet yield. Adding a central metal atom or, for instance, changing it from diamagnetic to paramagnetic can also change the properties of the PS significantly. Thus, protoporphyrin IX is a good PS which fluoresces rather well, while upon insertion of Fe2+ into the center of the macrocycle, both these phenomena are strongly suppressed, indicating a change to internal conversion as the predominant pathway.

Photochemistry

In the previous section, the photophysics of photosensitizers (PS) showed that short-lived singlet states (S1) and longer-lived metastable triplet states (T1) are produced following photon absorption. Direct reactions of substrate molecules ‘M’ with the short-

lived S1 state of the PS are probably not important, and even if S1 collides with oxygen, which may induce intersystem crossing (ISC) to give the triplet state T1, singlet oxygen would probably not be produced as this process is highly endothermic. This is because the singlet-triplet splitting in porphyrins is frequently of the order of 8 kcal/mole compared to the 1O2 excitation energy of nearly 23 kcal/mole.

The chemical reactions that are induced by the triplet state photosensitizer (T1) are summarized in Figure 6 (see also Jori and Spikes10).

The chemical reactions shown in Figure 6 follow three main pathways, which start from the common metastable triplet state of the photosensitizer (3PS):

(1) energy transfer, either to oxygen to give O2(1∆) which then either reacts with PS (bleaching it in a ‘cage’ reaction) or oxidizes a biomolecular target close to the PS. This is generally called a Type II reaction. Alternatively, the 3PS can transfer its energy to M. The second pathway: (2) hydrogen atom exchange between the PS and M leads to radical intermediates which may combine with oxygen to form peroxide radicals as subsequent intermediates leading to oxidized M. Finally, (3) electron transfer between 3PS and M leads to radical ions which together with O2 can lead to other oxidizing reactive intermediates, such as the superoxide radical anion (O2•-), H2O2 and the very reactive hydroxyl (OH•) radical. Processes (2) and (3) together are called Type I reactions. It should be noted that Type II reactions require oxygen in the first step, while Type I reactions only involve oxygen further downstream in the reaction mechanism. From the three pathways indicated in Figure 6, the energy transfer from 3PS to oxygen is generally envisaged to be one of the more important steps. This is shown in more detail in Figure 7 (see also, Turro11).

Finally, it should be noted that the energy transfer to M (3PS + 0M 0PS + 3M) is too endothermic for most biomolecules, β-carotene being a typical exception.

In a biological environment, singlet oxygen (1O2) either reacts rapidly or is quenched so that reactions take place at distances of only a few tens of nanometers from the location of the PS, so that PS localization determines where PDT damage takes place. Some typical reactions of 1O2 are shown in Figure 8.

Biomolecular pathway changes

In vivo, PDT effects are interpreted in terms of three mechanisms: cellular, vascular, and immunological effects. The relative contribution of each of these to the overall PDT efficacy in destroying a tumor

depends on the tissue, the sensitizer, and the conditions applied. The latter include the time between drug and light application and the drug delivery system, both of which determine drug localization at the time of light application. The quantities of drug and light also play a role. In this overview, we are particularly concerned with the vascular PDT ef- fect,1,2,5,12-16 which may be observed as a vasoconstriction directly upon light exposure or several hours later. Endothelial cells are supposedly the main target and, depending upon the drug, either mainly the mitochondria (this is the case for BPD-MA) or predominantly the lysosomes or the plasma membrane may be targeted, among others. Lysosomal localizing (water soluble) PSs tend to cause cell death by necrosis, or can lead to apoptosis via the release of cathepsins and caspase 3. On the other hand, mitochondrial localizing PSs mainly lead to cell death by apoptosis. In these organelles, PDT-induced lipid

peroxidation may lead, among other things, to organelle membrane disruption, change in membrane potential, or damage to membrane proteins. Apart from apoptosis and necrosis as a death response to PDT, partially damaged cells will show an activated rescue response in the form of release of heat shock proteins, glucose-regulated proteins and heme oxygenase (see, Xue et al.,17,18 Gomer et al.19-21 and Morgan et al.22). PDT also influences surface receptors and induces cytokines, which probably influence the immune response.

For an example of some of the known details of the biochemical apoptotic pathways induced by PDT, the reader is referred to Figure 9 and to Oleinick et al.23 and Roth and Reed.24

Damage by PDT may occur either mainly in the cell membrane, the mitochondria, or the lysosomes. We take the case of a PS (BPD-MA) mainly localized in the mitochondria at the time of light application, in some cases causing the loss of the mitochondrial membrane potential, possibly due to the opening of a large conductance channel called the mitochondrial permeability transition pore complex (PTPC). This can contribute to the release of cytochrome-c into the cytosol, where it forms a multi-protein complex called an apoptosome with apoptosis-activat- ing factor-1 (APAF-1) and ATP. The latter recruits and activates pro-caspase 9 via the caspase dimer. The name caspase comes from a cysteine protease acting on aspartic acid, i.e., a protein that cleaves another protein at a specific site. The apoptosome now releases caspase 9 which, in turn, induces caspase 3 release.

The latter is a key protein in apoptosis induction and causes the cleavage of multiple proteins, among which PARP (poly-ADP-ribose polymerase) and DNA-PK, which normally acts to repair DNA damage. Caspase 3 also attacks ICAD, thus releasing CAD which itself attacks DNA. Caspase 3 can also

activate pro-caspase 6, thereby releasing caspase 6 which cleaves the lamins of the cell nucleus, thus inducing nuclear breakdown. Many other substrates are attacked by caspase 3, among which SHREBs, Gelsolin, caspase 7, caspase 9, MDM2, GAS2, FODRIN, FAK, etc., all of which are involved in apoptosis. In a parallel process, the release of cyto- chrome-c can also indirectly influence the activity of all these caspases by the release of Smac/Diablo,2428 which interferes with the release of IAPs (inhibitor of apoptosis proteins). The latter controls the release of all these caspases.

For water-soluble PSs that might localize in lysosomes, PDT leads to the release of cathepsins which, among other things, help to release caspase 3. For the sake of simplicity, this has not been added to Figure 9.

It cannot be completely ruled out that, in the case of a vascular PDT effect following damage to endothelial cells at very short intervals after PS injection, cell surface receptors play a role. Activation of cell death receptors such as TRAIL (TNF-related apoptosis-inducing ligand), FAS, and TNFR1 (tumor necrosis factor receptor 1) via specific ligands will cause adaptor proteins such as FADD (FAS associated death domain) to bind to their cytosolic component. This complex then recruits and activates procaspase 8, causing caspase 8 release. The latter acts to give caspase 3. This picture is further complicated by the fact that proteins of the Bcl-2 family regulate apoptosis and contain a number of both pro-apoptotic members such as Bid and Bax (the latter may be able to form a transmembrane pore, leading to cytochrome-c release) as well as many anti-apoptotic members, among which Bcl-2 itself, as well as Bcl-xL which are located in the outer mitochondrial membrane and promote cell survival. Caspase 8 cleaves Bid in the cytosol, generating truncated Bid (t-Bid), which then relocates to the

mitochondrial membrane, thus helping to release cytochrome-c. It is hoped that a better understanding of these pathways will lead to more effective PDT via apoptosis, and hence possibly lower drug and light doses compared to necrosis. It may also be speculated that the selectivity of apoptosis induced by PDT might be easier to manipulate than that of necrotic cell death, so that improved selective destruction of certain tissues can be envisaged. More information on caspases can be found in Salvesen and Dixit,29 Cohen,30 Qin et al.,31 and Thornberry and Lazebnik.32

Vascular effects of photodynamic therapy

Photodynamic therapy with various photosensitizers under a specific set of applied conditions can have a predominant vascular component. This has led to the use of PDT for treating several non-can- cerous lesions such as atherosclerotic plaque, choroidal neovascularization (CNV) of the retina, re-stenosis after balloon angioplasty, and port-wine stains. Microvascular damage and blood flow stasis during and after PDT have consequently been the focus of a number of recent studies.1,2,12-15,33-35 Although many details still remain unknown, a certain picture illustrated by Figure 10, can be extracted from the available information. Early damage, after the i.v. injection of PS and illumination, is observed in endothelial cells and the sub-endothelium. In particular, PDT-induced changes to endothelial cells can be found on the luminal surface, in cytoplasmic microtubules, on cytoskeletal proteins, and in mitochondria. Endothelial cells undergoing PDT then retract and lose their tight junctions with adjacent cells, thus exposing the basement membrane as shown in Figure 10b. The latter leads to the activation of platelets and polymorphonuclear leukocytes which, in turn, causes the increased release of eicosanoids, and finally the aggregation of platelets on the exposed basement membrane of the vessel wall.

A further observation following PDT is the adhesion of polymorphonuclear leukocytes to the vessel

wall. A consequence of these changes is the release of biochemicals, including the eicosanoids thromboxane and leukotriene B4 and C4. It should be noted that the rounding of the endothelial cells combined with the biochemical changes first causes an increase in vessel permeability and leakage, before finally leading to blood flow stasis, as illustrated by Figure 10c.

The PDT-induced release of biochemicals causes disturbance of the normal equilibrium between aggregating and disaggregating behavior, as well as between vasoconstriction and vasodilatation processes. This imbalance results in increased smooth muscle cell activity and effective vasoconstriction, combined with platelet aggregation to form a plug, which is stabilized by fibrins.

In a parallel mechanism, PDT damage to membrane lipids can cause the release of arachidonic acid and a consequent biochemical cascade involving cyclooxygenase, prostaglandin endoperoxides, and finally also the production of the vasoconstrictor, thromboxane. The overall result of all this is blood flow stasis, which may to some extent have been aided by the increased interstitial pressure following enhanced leakage.

In order to investigate the effects of PDT on the blood flow in more detail, PDT-induced leakage and vessel constriction were studied in a CAM model (chicken embryo’s chorioallantoic membrane) in our laboratory. Some results obtained with this model using various PSs are shown below. In Figure 11, typical experimental results compare photosensitizer leakage from CNV in a human eye (on the left) with leakage from small CAM vessels (on the right). The leakage of PS is important in the PDT of CNV associated with age-related macular degeneration (AMD), since too much leakage of the PS before light application may result in photodynamic damage to parts of the retina close to the leaky neovessels that we want to preserve (such as, for instance, the photoreceptors). The two images on the left-hand side of Figure 11 show the strong localized leakage of fluorescein at short and at longer times after injection. This leakage is typical of aggressive ‘classical’ CNV, which in this case was later treated by PDT with

Visudyne®. On the right-hand side, in the CAM model, the fluorescence pharmacokinetics is shown for a water-soluble dye (Rhodamine 101) after i.v. injection,36 demonstrating the arrival of the fluorescing substance in different parts of the vasculature at short intervals after injection (top), and the leakage of the substance at longer intervals after injection (bottom). The basic idea behind this experiment is to establish a ‘leakage-scale-relationship’ between the human eye and the CAM model.

The efficacy of a PS in blood flow stasis is shown in Figure 12, once again comparing this property between the human eye (on the left) and the CAM vessels (on the right). The goal also being to screen new PSs for PDT of CNV in AMD and to establish a ‘PDT-blood-flow-stasis’ relationship between the clinical observations and the results in the preclinical model. Thus, on the left-hand side of Figure 12,

the leakage of fluorescein is shown in one patient, one week after PDT treatment with Visudyne®. The dark hypofluorescent spot that can be seen in the macular region shows, one week after PDT, both the disappearance of the CNV leakage that existed prior to PDT, and the decreased leakage of the partially closed choriocapillaries in this region. At the right top of Figure 12, the time frame of PDT with Visudyne® on CAM36 is shown by Visudyne® angiograms prior to PDT (40 sec), while light application (2 min) shows the treated area. At the right bottom of Figure 12, the blood flow stasis is shown 24 hours after PDT by a Rhodamine 101 angiogram. A hypofluorescent spot, quite similar to that shown in the clinical contest, indicative of blood flow stasis in the irradiated region, does not show immediately after PDT, but becomes clearly visible 24 hours after treatment.

Selectivity in photodynamic therapy

‘Selectivity’ in the PDT of tumors will depend on the drug, drug carrier, application of light, and tissue properties. Selectivity may be defined in many ways. Here, we present some possible reasons for ‘drug concentration-selectivity’ in PDT:

Selective uptake

Leaky neovessels in tumors can cause more of the drug to be delivered. This leakiness may be temporarily enhanced by PDT itself. Tumors may also have increased the activity of low density lipoprotein (LDL) receptors and/or other receptors, such as albumin. Hydrophobic sensitizers may be bound in the lipid core of lipoproteins, while hydrophilic PSs can be transported by albumin.

Selective retention

Tumor-associated macrophages may accumulate both aggregated lipophilic PSs and lipoproteins overloaded with PSs. pH-lowering advanced cancers can cause a decrease in the solubility of certain porphyrins. Reduced lymphatic drainage in neoplastic tissue may cause a build-up of certain molecules.

Furthermore, a PS may be targeted to some receptor, which is selectively expressed in a tumor or in tumor neovasculature. This may be done by attaching a PS covalently to a monoclonal antibody or antibody fragment, which is targeted to a specific antigen. Alternatively, the PS could be attached to a small peptide targeted to an integrin such as αvβ3, which has been shown to be expressed selectively on some tumor neovasculature.

The case of selectivity in the PDT of CNV associated with exudative AMD is discussed in detail by Birngruber in another chapter in this volume. Here, this topic is discussed briefly from a slightly different point of view.

Figure 12 showed a fluorescein angiogram of the macular region of a human eye after PDT. The irradiated zone shows up as a dark hypofluorescent region. This implies that, not only have the exudative neovessels been closed, but also the choriocapillaries have at least partially been closed. The retinal capillaries are clearly patent. Hence, the treatment is perfectly selective in the sense of sparing the retinal capillaries – which is all-important – and not completely selective in the sense of partially closing the choriocapillaries.

In the following section, we discuss the selectivity issues that apparently play a role in the PDT of CNV associated with AMD.

Figure 13 shows a schematic and simplified diagram of the central region of the retina, known as the macula. The light is shown coming in from the top. This activates the photoreceptors (there are mostly cones for high resolution and color vision in the macula). The signal from the cones is pre-

processed by bipolar and horizontal cells, amacrine cells, and ganglion cells (none shown) before being transferred to the brain by axons, which leave the eye in a bundle via the optic nerve. Below the photoreceptors is the retinal pigment epithelium (RPE), a layer of ‘macrophage-like’, highly pigmented cells. There are five types of blood vessels in the diagram. Near the surface (top) of the retina are the retinal arteries and veins, which feed the small and not very dense network of retinal capillaries, which surrounds the macula. In the macular region itself, there are no retinal vessels, as these would tend to blur the high-resolution image. Closure of the retinal capillaries in PDT must absolutely be avoided, since this is extremely damaging to the visual acuity. Below the RPE there is a zone called Bruch’s membrane, and below this are the choroidal capillaries, which are fed by the larger choroidal vessels. Although the etiology of exudative (neovascular) AMD is not completely understood, one reason for the growth of CNV from the choriocapillaries is probably a lack of oxygen and other nutrients at the level of the photoreceptors. The leaking CNV end up by disturbing visual acuity, as has been described in detail in the literature.37 The following question may now be asked: what selectivity is required from PDT? First, as described above, closure of the retinal capillaries must be avoided. This is probably facilitated by the lower partial pressure of oxygen in the retinal circulation compared to the choroidal circulation,38-41 the former being part of the blood-brain barrier associated vasculature. In fact, the human retina receives its oxygen from two separate sources: the retinal circulation is the main supply of O2 for the inner retina between the vitreal surface and the inner nuclear layer, while the choroidal circulation oxygenates the remainder of the retina and the RPE (see Fig. 13). Furthermore, oxygen consumption is not homogeneous across the retina. It is probably highest near the photoreceptor inner segments (IS in Fig. 13). Therefore, the non-uniformity of the oxygen supply and oxygen consumption in the retina must lead to oxygen gradients.40,42-45 On an even smaller scale, these gradients may be further disturbed by the differences in oxygen solubility in different tissue compartments. Thus, O2 is less soluble in the cytosol than in the plasma membrane.

Tighter contacts between endothelial cells in the retinal circulation may also render PDT relatively less effective. A second type of selectivity is that

PDT should, at least to some extent, be restricted to the vasculature. This means that PSs should not be allowed to leak significantly out of the CNV on the timescale of the treatment as it is important to avoid damage to the photoreceptors and the neural retina, as well as major damage to the RPE. The RPE cells probably pick up leaked PSs rapidly. And even though some damage to the RPE (which might lead to replacement of ‘aged’ RPE cells) might be of interest, major damage to the RPE should probably

be avoided. A third type of selectivity implies that we want to close the CNV without destroying the choriocapillaries or larger choroidal vessels. The larger choroidal vessels may be protected by a significantly different amount of collagen in the vessel walls compared to the smaller diameter vessels. It has been pointed out by Hasan et al.2 that neovasculature in some diseases tends to have enhanced activity of receptors for LDL and albumin. Thus, lipophilic substances such as BPD-MA attached to LDL may be preferentially taken up in the CNV. Some proof of enhanced BPD-MA in the neovascular region can be seen in Figure 14, which shows the fluorescence pharmacokinetics of BPD-MA in the eye fundus at intervals up to 30 minutes.

Analysis of these images46 indicates a possible degree of selectivity of BPD-MA for the CNV. Nevertheless, PDT also leads to at least temporary closure of a significant part of the choriocapillaries, as can be seen from the hypofluorescent spot in the fluorescence angiograms obtained at early and late time intervals with both ICG and fluorescein (Fig. 12). The hypofluorescence observed with both compounds indicates a significant closure of the choriocapillaries one week after PDT. Thus, the desired selectivity between closing CNV and not closing choriocapillaries is incomplete. In order to attain this selectivity, different approaches are taken, two of which involve the increased expression of αvβ3 integrins at the surface of neovascular endothelial cells.

Birchler et al.47 from Zurich have tried to link a PS to an antibody fragment that attaches to fibronectin, which itself attaches to αvβ3. Other groups, including ourselves, are presently trying to attach PS to RGD-peptide-related structures which attach directly to αvβ3. Only the future will show whether improved PDT selectivity can be attained with such approaches, and whether this selectivity will significantly influence the treatment efficacy. Non-closure of the choriocapillaries should increase tissue oxygenation after PDT, and decrease PDT-related inflammation, both of which should reduce the recanalization/regrowth of CNV that hampers present-day treatments and makes repeat treatments necessary. Finally, of course, it should be mentioned that, with PDT of CNV in AMD, we treat the effects of the disease but not its cause(s).

Conclusions

In this overview, we have described some of the basic photophysics, photochemistry, and photobiology associated with PDT. A simplified mechanism of the biological cascades leading to apoptosis has also been described, as well as details of PDT in vessels leading to blood flow stasis. Finally, selectivity issues in the PDT of exudative AMD are discussed.

Acknowledgment

We would like to thank N. Lange for the preparation of Figures 11 and 12.

References

1.Van den Bergh H: Photodynamic therapy of age related macular degeneration: history and principles. Semin Ophthalmol 25: 2002 (accepted for publication)

2.Hasan T et al: Photodynamic therapy of cancer. In: Holland JF et al (eds) Cancer Medicine, pp 489-502. Hamilton, BC: Decker Inc 2000

3.Milgrom LR: The Colours of Life: An Introduction to the Chemistry of Porphyrins and Related Compounds. New York, NY: Oxford University Press Inc 1997

4.Bonnett R: Chemical Aspects of Photodynamic Therapy. New York, NY: Gordon and Breach Sci Publ 2000

5.Henderson BW, Dougherty TJ: Photodynamic Therapy: Basic Principles and Clinical Applications. New York, NY: M Dekker 1992

6.Van den Bergh H: On the evolution of some endoscopic light delivery systems for photodynamic therapy. Endoscopy 30:392-407, 1998

7.Monnier P et al: Photodetection and photodynamic therapy of ‘early’ squamous cell carcinomas of the pharynx, oesophagus and tracheo-bronchial tree. Lasers Med Sci 5:149-169, 1990

8.Monnier P et al: Further appraisal of PDI and PDT of early squamous cell carcinomas of the pharynx, oesophagus and bronchi. In: Spinelli P et al (eds) Photodynamic therapy and biomedical lasers, pp 7-14. Amsterdam: Elsevier Sci Publ 1992

9.Radu A et al: Photodynamic therapy for 101 early cancers of the upper aerodigestive tract, the esophagus, and the bronchi: a single-institution experience. Diagnost Therapeut Endoscop 5:145-154, 1999

10.Jori G, Spikes D: Topics in photomedicine. In: SmithKendric C (ed) Topics in Photomedicine, pp 183-318. Cambridge: Perseus Publ 1984

11.Turro NJ: Modern Molecular Photochemistry. University Science Books, Sausalito, CA: 1991

12.Fingar VH et al: The role of microvascular damage in photodynamic therapy: the effect of treatment on vessel constriction, permeability, and leukocyte adhesion. Cancer Res 52:4914-4921, 1992

13.Fingar VH: Vascular effects of photodynamic therapy. J Clin Laser Med Surg 14:323-328, 1996.

14.Fingar VH et al: Analysis of acute vascular damage after photodynamic therapy using benzoporphyrin derivative (BPD). Br J Cancer 79:1702-1708, 1999.

15.Krammer B: Vascular effects of photodynamic therapy. Anticancer Res 21:4271-4277, 2001

16.Star WM et al: Destructive effect of photoradiation on the microcirculation of a rat mammary tumor growing in ‘sandwich’ observation chambers. Prog Clin Biol Res 170:637645, 1984

17.Xue LY et al: Elevation of GRP-78 and loss of HSP-70 following photodynamic treatment of V79 cells: sensitization by nigericin. Photochem Photobiol 62:135-143, 1995

18.Xue LY et al: Rapid tyrosine phosphorylation of HS1 in the response of mouse lymphoma L5178Y-R cells to photodynamic treatment sensitized by the phthalocyanine Pc 4. Photochem Photobiol 66:105-113, 1997

19.Gomer CJ et al: Photodynamic therapy-mediated oxidative stress can induce expression of heat shock proteins. Cancer Res 56:2355-2360, 1996

20.Gomer CJ et al: Glucose regulated protein induction and cellular resistance to oxidative stress mediated by porphyrin photosensitization. Cancer Res 51:6574-6579, 1991

21.Gomer CJ et al: Increased transcription and translation of heme oxygenase in Chinese hamster fibroblasts following photodynamic stress or Photofrin II incubation. Photochem Photobiol 53:275-279, 1991

22.Morgan J et al: GRP78 induction by calcium ionophore potentiates photodynamic therapy using the mitochondrial targeting dye victoria blue BO. Photochem Photobiol 67:155164, 1998

23.Oleinick NL et al: The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 1:1-21, 2002

24.Roth W, Reed JC: Apoptosis and cancer: when BAX is TRAILing away. Nat Med 8:216-218, 2002

25.Reed JC: Apoptosis-based therapies. Nature Rev Drug Disc 1:111-121, 2002

26.Nicholson DW: From bench to clinic with apoptosis-based therapeutic agents. Nature 407:810-816, 2000

27.LeBlanc H et al: Tumor-cell resistance to death receptorinduced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax. Nat Med 8:274-281, 2002

28.Deng Y et al: TRAIL-induced apoptosis requires Bax-de- pendent mitochondrial release of Smac/DIABLO. Genes Dev 16:33-45, 2002

29.Salvesen GS, Dixit VM: Caspases: intracellular signaling by proteolysis. Cell 91:443-446, 1997

30.Cohen GM: Caspases: the executioners of apoptosis. Biochem J 326:1-16, 1997

31.Qin H et al: Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1. Nature 399:549557, 1999

32.Thornberry NA, Lazebnik Y: Caspases: enemies within. Science 281:1312-1316, 1998

33.Star WM et al: Destruction of rat mammary tumor and normal tissue microcirculation by hematoporphyrin derivative photoradiation observed in vivo in sandwich observation chambers. Cancer Res 46:2532-2540, 1986

34.Henderson BW et al: Effects of photodynamic treatment of platelets or endothelial cells in vitro on platelet aggregation. Photochem Photobiol 56:513-521, 1992

35.Henderson BW, Dougherty TJ: How does photodynamic therapy work? Photochem Photobiol 55:145-157, 1992

36.Lange N et al: A new drug-screening procedure for photosensitizing agents used in photodynamic therapy for CNV. Invest Ophthalmol Vis Sci 42:38-46, 2001

37.Schmidt-Erfurth U et al: Photodynamic therapy of subfoveal choroidal neovascularization: clinical and angiographic examples. Graefe’s Arch Clin Exp Ophthalmol 236:365-374, 1998

38.Yu DY, Cringle SJ: Oxygen distribution and consumption within the retina in vascularised and avascular retinas and in animal models of retinal disease. Prog Retin Eye Res 20:175-208, 2001

39.Tornquist P, Alm A: Retinal and choroidal contribution to retinal metabolism in vivo: a study in pigs. Acta Physiol Scand 106:351-357, 1979

40.Tsacopoulos M et al: Studies on retinal oxygenation. In:

Grote J et al (eds) Oxygen Transport to Tissue II, pp 413-

416. New York, NY: Plenum Publ Corp 1976

41.Pournaras CJ: Retinal oxygen distribution: its role in the physiopathology of vasoproliferative microangiopathies. Retina 15:332-347, 1995

42.Drujan BD, Svaetichin G: Characterization of different classes of isolated retinal cells. Vision Res 12:1777-1784, 1972

43.Linsenmeier RA: Effects of light and darkness on oxygen distribution and consumption in the cat retina. J Gen Physiol 88:521-542, 1986

44.Dollery CT et al: Oxygen supply to the retina from the retinal and choroidal circulations at normal and increased arterial oxygen tensions. Invest Ophthalmol 8:588-594, 1969

45.Alder VA et al: The retinal oxygen profile in cats. Invest Ophthalmol Vis Sci 24:30-36, 1983

46.Sickenberg M et al: A computer-based method to quantify the classic pattern of choroidal neovascularization in order to monitor photodynamic therapy. Graefe’s Arch Clin Exp Ophthalmol 237:353-360, 1999

47.Birchler M et al: Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment. Nat Biotechnol 17:984-988, 1999

Introduction

One of the essential advantages of the principle of photodynamic therapy is the possibility of using photosensitizer molecules which preferentially get attached to or incorporated into the desired target structure, in order to achieve optimal selectivity of the photodynamic action after light activation.1 Several targeting mechanisms and sensitizer distribution modalities are presently being evaluated for a variety of different PDT applications. In order to treat neovascular structures, a specific targeting process leading to the preferential uptake of the photosensitizer in new vessels has to be developed. An ultimate selectivity seems to be particularly necessary if the neovascularization is situated directly adjacent to functionally important structures, such as in the clinically important pathology of macular choroidal neovasculature (CNV). This chapter describes the concept of selective targeting proliferating vascular endothelial cells and reviews the experimental studies that eventually led to a new treatment modality for CNV-related macular diseases, such as age-related macular degeneration, pathological myopia, and other chorioretinal diseases.

Ocular PDT

The principle of PDT was first applied in ophthalmology in the 1980s by Sery,2 Gomer et al.,3 and Murphree et al.4 in order to investigate the potential for treating intraocular tumors. In all those investigations, hematoporphyrin derivate (HPD), the first commercially-available photosensitizer, was used. However, the selectively and efficiency of tumor

destruction, as well as the debridement capacity of the eye, were not high enough to lead to a successful new treatment modality. In the late 1980s, a number of papers were published that evaluated the possibilities of treating ocular neovascularizations photodynamically.5-9 Different photoactive substances, such as dehematoporphyrin ether, phthalocyanin and rose bengal were used with HPD. All these investigations showed thrombosis due to photo- dynamically-induced intraluminal action in normal retinal vessels,7 iris neovasculature,5 and experi- mentally-induced CNV in monkeys.6,8,9 These investigations demonstrated different degrees of efficacy and selectivity ranging from ‘PDT-augmented laser coagulation’6 to ‘closure of CNV with retinal preservation’,8 depending on the type of photosensitizers treatment protocol and dosimetry used in the experiments.

Vascular selectivity of photodynamic therapy

The rational approach for optimizing vascular PDT is to systematically search for the right kind of sensitizer with maximum affinity to vascular structures, investigate the optimal drug-light interval with the highest concentration gradient of the photosensitizer in the neovascular structures, and evaluate the appropriate drug and light dosimetry, avoiding systemic side-effects by applying the minimum drug dose necessary, and eliminating undesired unspecific thermal collateral damage by using low light irradiances for the photodynamic drug excitation. Therefore, the optimal compound would be a highly phototoxic sensitizer which selectively binds to vascular structures. In general, it is known that hydrophillic molecules have the tendency to be preferentially taken

up by the vasculature. Moreover, numerous experimental studies have shown that damage to the endothelial cell layer of the inner vessel wall – be this mechanical10 or thermal due to diathermia11 or argonlaser irradiation12 – leads to intravascular thrombosis due to thrombocyte agglutination with associated platelet and erythrocyte clumping.13,14 Therefore, the concept of selective vascular photodynamic therapy was to maximize photosensitizer uptake in the vascular endothelial cells. This specific targeting of endothelial cells can be mastered by complexing a potent photosensitizer with a carrier molecule with a high affinity to endothelial cells. Serum lipoproteins are promising candidates for such carrier systems, with their receptor-mediated internalization into endothelial cells.15

The therapeutic principle of neovascular selectivity

The clinical goal of neovascular photodynamic therapy is to treat neovascular diseases – most importantly CNV membranes and other chorioretinal vascular neoplasias such as choroidal and retinal hemangiomas – by a mechanism of action ultimately selective to the neovascular vessel wall. Consequently, the concept of selective vascular targeting could be even more specifically adapted to neovasculature if the carrier system were to be preferentially taken up by neovascular endothelial cells. Studies on tumor selectivity in photodynamic therapy have indicated that the uptake of lipophilic sensitizers in tumorous neovasculature is mediated by low density lipoprotein (LDL).16

Based on this argumentation, we hypothesized for the first time on the possibility of selective photothrombosis of the ocular neovasculature, using a sensitizer LDL complex for the selective targeting of proliferating neovascular endothelial cells.17,18,43 The desired selectivity is thought to be due to the increased LDL receptor expression in proliferating cells.44 In addition, improved efficacy might be achieved by the fact that the sensitizer LDL complex is internalized by endocytosis, causing enzymatically enhanced endothelial cell destruction after photoexcitation.19 The concept of selective neovascular photothrombosis is outlined in Figures 1a-g: a CNV membrane originating from the choriocapillaris proliferates through Bruch’s membrane into the

subretinal and retinal spaces (Fig. 1b). The new vessels (Fig. 1c), with their heavily leaking vessel walls, cause exudation and hemorrhages into the outer retinal space, eventually leading to the formation of a fibrovascular scar associated with a substantial loss of neural tissue (Fig. 1d). By applying a neovascular selective photosensitizer, the proliferating endothelial cells of the neovascular structures will be targeted (Fig. 1e), and after light application, the activated sensitizer will produce enough toxic singlet oxygen to selectively damaging the neovascular structure (Fig. 1f). The reactive intravascular thrombosis will then occlude the new vascular tissue without damaging the neural retina and retinal pigment epithelium, which should be able to re-establish the anatomical outer retinal structure (Fig. 1g). The assumed specific binding of the photosensitizer to LDL in the intravascular system is illustrated schematically in Figure 1h.

Experimental studies

The first experimental validation of the concept of the preferential uptake of the photosensitizer LDL complex in the ocular neovasculature, and consequently of selective thrombotic occlusion of the neovascular structures with minimal collateral photodynamic damage to the surrounding tissues, was performed in experimental models of the corneal neovasculature18 and tumor-induced choroidal neovascularization17 in rabbits.

Benzoporphyrin derivative monoacide (BPD-MA)

– currently available commercially in liposomal formulation under the name Verteporfin™ – was used either in liposomal formulation or was complexed with LDL before intravenous injection. Allison et al.20 and Richter et al.19 have shown that BPD in liposomal formulation injected into the blood circulation forms BPD-LDL complexes similar to those obtained extracorporeally. In corneal neovasculature, we were able to demonstrate that liposomal BPD and directly complexed BPD-LDL showed comparable vascular selectivity, and that subsequent photoactivation induced the same vascular effects.18 Figure 2 shows the chemical structure and action spectrum of BPD-MA. The uptake of liposomal BPD in the corneal vasculature, measured using laser-induced fluorescence (LIF) at different time intervals after drug injection, clearly demonstrates the preferential

Fig. 2. The chemical structure of the two regio-isomers of BPD-MA and the action spectrum of the photosensitizer. Photoactivation is performed with diode laser light around the peak wavelength of 689 nm.

Fig. 3. Uptake and retention of LDL-complexed BPD-MA in the corneal neovasculature of rabbit eyes measured over 48 hours by laser-induced fluorescence.

binding to neovascular structures. The normalized relative BPD fluorescence measured at a wavelength of 696 nm was excited at 337 nm, using a pulsed nitrogen laser.18 Figure 3 shows the kinetics of uptake and retention with maximum sensitizer accumulation at about one hour post-injection. The retention of the sensitizer shows exponential decay with a halflife time of about six hours, which is much longer than its life time in the blood serum. This rapid accumulation and retention suggest a short drug-light interval for the effective photodynamic activation of BPD. The pronounced maximum of the action spectrum at an infrared wavelength of 694 nm enables good light transmission through the optical media of the eye, even with moderately cataractous lenses, and provides enough light penetration to reach chorioretinal neovascularizations below the retinal pigment epithelium (RPE) and/or hemorrhagic exudation. The photothrombotic effect in the neovasculature after light activation of BPD-MA is demonstrated in Figures 4a-d. In this example, the liposomal LDL sensitizer with a BPD dose of 2 mg/ kg body weight was administered intravenously into Dutch belted rabbits. One hour post-injection, laser irradiation at a wavelength of 694 nm and an irra-

a.

b.

c.

d.

Figs. 4a-d. Photothrombotic effects in the corneal neovasculature of a rabbit eye. a,b: Color photographs of a corneal neovascular structure induced by mechanical irritation with intrastromal silk sutures before (a) and several minutes after (b) PDT, with light doses of 10 J/cm2 (arrow) and 25 J/cm2 (double arrow), respectively. c,d: Fluorescein angiography of the same eye before and after PDT. The neovascularizations are completely occluded in both treated areas.

Fig. 6. Light micrographs of a Green’s melanoma planted into the choroid of a rabbit one hour post-PDT. The endothelial cells of the tumor vessels show hyperchromatic cell nuclei as a typical sign of cell damage. The lumina of the vessels are completely thrombosed.

Fig. 7. Electron micrograph of a vessel from an implanted iris tumor in a rabbit eye two hours post-PDT. The primary damage to the vascular endothelial cells can be clearly seen: cytoplasmatic extrusion into the vessel lumen (arrows) indicates total cell destruction.

diance of 100 mW/cm2 was performed using light doses of 10 J/cm2 and 25 J/cm2, respectively.

Photographic documentation (Figs. 4a,b) and fluorescein angiography (Figs. 4c,d) demonstrate the structure and function of the neovasculature before (Figs. 4a,c) and several minutes after (Figs. 4b,d) photoactivation. Histological preparation shows the morphology of photodynamically-induced vessel occlusion. Light microscopy of semi-thin histological

preparations one hour after PDT clearly shows complete thrombotic occlusion of the corneal neovasculature (Fig. 5b) in contrast to the completely perfused neovasculature prior to PDT (Fig. 5a).

Further investigations into neovascular structures in experimental tumor models showed similar results after PDT with BPD-LDL complexes.17 One hour post-treatment, complete thrombosis of the tumor vasculature in Green’s melanomas implanted into the iris and choroid of rabbits was observed ophthalmoscopically and fluorescein-angiographically. No signs of recanalization of the occluded vessels were observed during the follow-up of up to three months. Light and electron microscopic evaluation of the treated vessel walls elucidated the mechanisms of thrombosis formation: as can be seen in Figure 6, one hour post-PDT, the endothelial cells are damaged and have substantial hypercromatic cell nuclei. The dilated vessel lumina are thrombosed. No collateral damage can be seen light microscopically in the adjacent tissue structures. Figure 7 shows the endothelial cell damage in greater detail in a higher resolution electron micrograph of an iris tumor vessel two hours post-PDT. The endothelial cell membranes have been disrupted and cytoplasm has extruded into the vascular lumen.

All these experimental findings strongly support our proposed concept of the photodynamically-gen- erated selective occlusion of neovascular structures: the primary photo-oxidative damage to the vascular endothelial cells induces thrombus formation by intravascular platelet activation and thrombocyte aggregation.

The photodynamically-generated, so-called photothrombosis of neovasculatures demonstrated here, with its spatial confinement to the inner structures of the vessel walls and their lumina, stimulated further animal studies,21,22 mainly in order to establish optimal drug and light dosimetry. It also led to the first clinical evaluation of the photodynamic treatment of CNV membranes in age-related macular de- generation,23-28 and other chorioretinal pathologies.29-31 The results of extended multicenter clinical trials of

the photodynamic therapy of choroidal neovascular membranes using Verteporfin™, and the current status of clinical experience, will be reviewed in the following chapter.32

Photosensitizers

The experimental proof of the principle of the selective photodynamic therapy of neovascular structures, as well as the first establishment of this therapeutic modality for the treatment of CNV, was carried out using BPD-MA as photosensitizer complexed with LDL or a liposomally formulated (Verteporfin™). The huge success of this therapeutic approach and the encouraging experimental and clinical results,5- 9,23-31,43 led to a number of further investigations using different photosensitizer compounds for the treatment of ocular neovascularities.

More than ten new photosensitizers were experimentally tested for neovascular treatment in the eye: SnET2,33 Lutex,34 ATX S10,35 NPe6,36 ICG,37 Lambda 27,38 Hypocrellin A,39 MV 6401,40 and Tookad.41 Since NPe6 and ICG are hydrophillic compounds and Lambda 27, Hypocrellin A, and MV 6401 are lipophilic dyes, only ATX S10 has amphyphillic properties with a measurable affinity to lipid membranes and, at the same time, the advantage of water solubility.42 Practically all these sensitizers are designed to have a strong light absorption band in the near-infrared region (between 660 and 780 nm), in order to provide rather undisturbed ocular light transmission, even in eyes with mild cataract, and to facilitate enough light penetration through the hemorrhages and pigmented layers masking the neovasculature.

The vessel closure experiments in rats, rabbits, and monkeys always followed the same principle: intravenous drug application was followed by light exposure of the vascular structure at the fundus of the animal eyes. Fluorescein angiography and histopathological examinations were performed in order to investigate the vessel closure and collateral damage in the treated areas. In most cases, variation of the drug and light dose and the drug-light interval resulted in clinically useful treatment parameters. A scientifically-based comparison between the different sensitizers is difficult at this point because of the very different stages of the various investigations. In general, the initial photodynamic effect with all sensitizers is damage to the vascular endothelium, which is then followed by intravascular thrombosis. However, the efficacy of this vessel closure mechanism and the side-effects in the retinal and choroidal vasculature, as well as the collateral damage in the neural retina and the retinal pigment epithelium, vary substantially for each sensitizer.

Two of the newly-investigated sensitizers were also used in clinical trials to treat CNV in age-related macular degeneration: tin ethyletiopurpurin (SnET2; Purlitin™) and lutetium texaphyrin (Lutex). Generally speaking, in both these sensitizers, the pre-

clinical experiments to validate the principle of intravascular thrombogenesis and to evaluate realistic treatment parameters, as well as the treatment protocols for the phase I/II and III clinical trials, were similar to the strategy originally used with the photosensitizer Verteporfin™. It is worth mentioning that almost all experimental studies, including those with new sensitizers not yet clinically evaluated, produced comparable results in terms of drug and light dosimetry, therapeutic mechanism, and time course for closure of the neovascular choroidal structures. Drug doses of 1-2 mg/kg body weight were applied systemically, and light doses of 20-50 J/cm2 were necessary to achieve vessel closure in the choroidal structures of rabbits and monkeys. Collateral damage in the RPE and neurosensory retina could be identified histologically, but this was small, thus indicating a substantial selectivity to vascular structures. These experimental findings were essentially consistent with those obtained with the liposomally formulated BPD-MA (Verteporfin™), a somewhat unexpected result due to the different biochemical properties of the sensitizers used. Verteporfin™ is a highly lipophilic compound in a liposomal formulation, Purlitin™ is also lipophilic, but is formulated in an emulsion to become water-soluble, and Lutex is hydrophillic in itself. These various properties should result in different drug kinetics in the ocular structures because of different uptake and retention in the tight retinal and in the fenestrated choroidal vascularization. However, the experimental results seemed to show a rather uniform damage profile in the vasculature, even with very different retention times in the skin, ranging from weeks (Purlitin™) to several hours (Verteporfin™). The clinical trials with Purlitin™ (phase III) and Lutex (phase I/II), were terminated before study reports had been published. Treatment efficacy as well as systemic and ocular side-effects were obviously not satisfactory, at least in comparison with the approved sensitizer, Verteporfin™. In contrast to the experimental findings, the clinical results indicate that efficacy and biodistribution of the sensitizers play an important role in the required selectivity of the ocular neovasculature, where new vascular structures have to be treated in the immediate vicinity of the anatomical resting retinal and large choroidal vasculature, and where functionally important tissues, such as the neurosensory retina, as well as extremely active phagocytic structures, such as the RPE, must remain undamaged. Further large scale research and studies will be required to establish new sensitizers for the treatment of neovascular structures in ocular tissue.

Conclusions

The concept of selective vascular PDT as the treatment principle for neovascular diseases in the eye was proven in animal experiments. Corneal neovas-

cularizations as well as neovascular structures in chorioidal tumors were selectively thrombosed using the photosensitizer benzoporphyrine derivative monoacid ring A (BPD-MA) complexed with low density lipoproteins (LDL). The LDL-formulation was achieved experimentally either by covalently binding BPD-MA with LDL before intravenous injection or by liposomal delivery of BPD-MA. The selectively targeted neovascular structures showed damage of the intravascular endothelial layer right after photodynamic light activation. This damage was then followed by intravascular thrombosis.

The therapeutic value of the achieved photothrombosis was successfully evaluated in clinical trials. The current status of the potential value and the limitations of this new treatment modality is described in a separate chapter of this book.32

After the first successful clinical trials numerous other photosensitizers have been investigated for vascular PDT in the eye both experimentally as well as clinically. Despite of experimentally encouraging results no photosensitizer other than BPD (Visudyne) has passed prospective clinical trials to date.

References

1.Henderson BW, Dougherty TJ: How does photodynamic therapy work? Photochem Photobiol 55:145-157, 1992

2.Sery TW: Photodynamic killing of retinoblastoma cells with hematoporphyrin derivative and light. Cancer Res 39:96100, 1979

3.Gomer CJ, Dorion DR, White L, Jester JV, Dunn S, Szirth BC, Razum NJ, Murphree AL: Hematoporphyrin derivative photoradiation induced damage to normal and tumor tissue of the pigmented rabbit eye. Curr Eye Res 3:229-237, 1984

4.Murphree AL, Cote M, Gomer CJ: The evolution of photodynamic therapy techniques in the treatment of intraocular tumors. Photochem Photobiol 46:919-923, 1987

5.Packer AJ, Tse DT, Yuon-Qing G, Hayreh SS: Hematoporphyrin photoradiation therapy for iris neovascularization. Arch Ophthalmol 102:1193-1197, 1987

6.Thomas EL, Langhofer M: Closure of experimental subretinal neovascular vessels with hematoporphyrin-ether augmented argon green laser photocoagulation. Photochem Photobiol 46:881-886, 1987

7.Nanda SK, Hatchell DL, Tiedeman JS, Dutton JJ, Hatchell M, McAdoo T: A new method for vascular occlusion. Arch Ophthalmol 105:1121-1124, 1987

8.Kilman GH, Stern D, Gregory WA: Angiography and photodynamic therapy of experimental choroidal neovascularization using phthalocyanin dye. Invest Ophthalmol Vis Sci 30:S371, 1989

9.Miller H, Miller B: Photodynamic therapy of subretinal neovascularization in the monkey eye. Arch Ophthalmol 111:855-860, 1993

10.Honour AJ, Mitchell JRA: Platelet clumping in injured vessels. Br J Exp Pathol 45:75-87, 1964

11.Callahan AB, Lutz BR, Fulton GP, Dengelmann I: Smooth muscle and thrombus thresholds to unipolar stimulation of small blood vessels. Angiology 2:35-39, 1969

12.Boergen KP, Birngruber R, Hillenkamp F: Laser-induced endovascular thrombosis as a possibility of selective vessel closure. Ophthalmic Res 13:139-150, 1981

13.Baumgartner HR: Platelet interaction with vascular structures. Thromb Diath Haemorrh 51:151-176, 1972

14.Ashford TB, Freiman DG: The role of the endothelium in the initial phases of thromboses. Am J Pathol 50:257-273, 1967

15.Jori G, Reddi E: The role of lipoproteins in the delivery of tumor-targeting photosensitizers. Int J Biochem 25:13691375, 1993

16.Roberts WG, Hasan T: Role of neovasculature and vascular permeability on the tumor retention of photodynamic agents. Cancer Res 52:924-930, 1992

17.Schmidt-Erfurth U, Baumann W, Gragoudas E, Flotte TJ, Michaud NA, Birngruber R, Hasan T: Photodynamic therapy of experimental choroidal melanoma using lipoproteindelivered benzoporphyrin. Ophthalmology 101(1):89-99, 1994

18.Schmidt-Erfurth U, Hasan T, Schomacker K, Flotte TJ, Birngruber R: In vivo uptake of liposomal benzoporphyrin derivative and photothrombosis in experimental corneal neovascularization. Lasers Surg Med 17:178-188, 1995

19.Richter AM, Waterfield E, Jain AK, Canaan AJ, Allison BA, Levy JG: Liposomal delivery of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD), to tumor tissue in a mouse tumor model. Photochem Photobiol 57:1000-1006, 1993

20.Allison BA, Pritchard PH, Levy JG: Evidence for low-density lipoprotein receptor-mediated uptake of benzoporphyrin derivative. (Published erratum appears in Br J Cancer 71(1):214, 1995) Br J Cancer 69:833-839, 1994

21.Miller JW, Walsh AW, Kramer M, Hasan T, Michaud N, Flotte TJ, Haimovici R, Gragoudas ES: Photodynamic therapy of experimental choroidal neovascularization using lipoprotein-delivered benzoporphyrin. Arch Ophthalmol 113:810-818, 1995

22.Kramer M, Miller J, Michaud N, Moulton RS, Hasan T, Flotte TJ, Gragoudas ES: Liposomal BPD verteporfin photodynamic therapy: selective treatment of choroidal neovascularization in monkeys. Ophthalmology 103:427-438, 1996

23.Schmidt-Erfurth U, Miller W, Sickenberg M, Bunse A, Laqua H, Gragoudas E, Zofragos L, Birngruber R, Van den Bergh H, Strong A, Manjuris U, Fsadi M, Lane AM, Piguet B, Bressler NM: Photodynamic therapy of subfoveal choroidal neovascularization: clinical and angiographic examples. Graefe’s Arch Clin Exp Ophthalmol 236:365-374, 1998

24.Miller JW, Schmidt-Erfurth U, Sickenberg M, Pournaras CJ, Laqua H, Barbazetto I, Zografos L, Piguet B, Donati G, Lane AN, Birngruber R, Van den Bergh H, Strong A, Manjuris U: Photodynamic therapy with Verteporfin for choroidal neovascularization caused by age-related macular degeneration: results of a single treatment in a phase 1 and 2 study. Arch Ophthalmol 117:1161-1173, 1999

25.Schmidt-Erfurth U, Miller JW, Sickenberg M, Laqua H, Barbazetto I, Gragoudas ES, Zografos L, Piguet B, Pournaras CJ, Donati G, Lane AN, Birngruber R, Van den Bergh H, Strong A, Manjuris U, Gray T, Fsadni M, Bressler NM: Photodynamic Therapy with Verteporfin for choroidal neovascularization caused by age-related macular degeneration: results of retreatment in a phase 1 and 2 study. Arch Ophthalmol 117:1177-1187, 1999

26.Photodymamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with Verteportin: one-year results of 2 randomized clinical trials: treatment of age-related macular degeneration with photodynamic therapy (TAP) study group: TAP report 1. Arch Ophthalmol 117:1329-1345, 1999

27.Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with Verteporfin:

two-year results of 2 randomized clinical trials: TAP report 2. Arch Ophthalmol 119:198-207, 2001

28.Verteporfin therapy of subfoveal choroidal neovascularization in age-related macular degeneration: two-year results of a randomized clinical trial including lesions with occult with no classic choroidal neovascularization: VIP report 2. Am J Ophthalmol 131:541-560, 2001

29.Sickenberg M, Schmidt-Erfurth U, Miller JW, Pournaras CJ, Zografos L, Piquet B, Donati G, Laqua H, Barbazetto I, Gragoudas ES, Lane A-M, Birngruber R, Van den Bergh H, Strong HA, Manjuris U, Gray T, Fsadni M, Bressler NM: A preliminary study of photodynamic therapy using Verteporfin for choroidal neovascularization in pathologic myopia, ocular histoplasmosis syndrome, angioid streaks, and idiopathic causes. Arch Ophthalmol 117:327-336, 2000

30.Photodynamic therapy of subfoveal choroidal neovascularization in pathologic myopia with Verteporfin: oneyear results of a randomized clinical trial: VIP report 1. Ophthalmology 108:841-852, 2001

31.Barbazetto I, Schmidt-Erfurth U: Photodynamic therapy of choroidal hemangioma: two case reports. Clin Exp Ophthalmol 238:214-221, 2000

32.Schmidt-Erfurth U, Michels S: Photodynamic Therapy: clinical status. This volume.

33.Peyman GA, Moshfeghi DM, Moshfeghi A, Khoobehi B, Doiron DR, Primbs GB, Crean DH: Photodynamic therapy for choriocapillaris using tin ethyl etiopurpurin (SnET2). Ophthalmic Surg Lasers 28:409-419, 1997

34.Arbour JD, Connolly EJ, Graham K, Carson D, Michaud N, Flotte T, Gragoudas ES, Miller JW: Photodynamic therapy of experimental choroidal neovascularization in a monkey model using intravenous infusion of lutetium texaphyrin. Invest Ophthalmol Vis Sci 40:S401, 1999

35.Obana A, Goto Y, Kaneda K, Nakajima S, Takemura T, Miki T: Selective occlusion of choroidal neovascularization by photodynamic therapy with a water-soluble photosensi-

tizer, ATX-S10. Lasers Surg Med 24:209-222, 1999

36.Kazi AA, Peyman A, Unal M, Knoobehi B, Yoneya S, Mori Keisuke, Moshfeghi D, Moshfeghi AA: Threshold power levels for Npe6 photodynamic therapy. Ophthalmic Surg Lasers 31:136-142, 2000

37.Costa RA, Farah ME, Morales PH, Freymuller E, Smith R, Cardillo JA: Choriocapillaris photodynamic therapy using indocyanine green. Invest Ophthalmol Vis Sci 42:S438, 2001

38.Kazi AA, Peyman GA, Genaidy M, Farahat HG, Morgan A, Bisland S: Evaluation of threshold parameters for choroidal vessel closure with the new photosensitizer Lambda 27. Invest Ophthalmol Vis Sci 42:S437, 2001

39.Grinstead LR Jr, Khoobehi B, Peyman GA, Passos E: Experimental photodynamic effects of hypocrellin A on the choriocapillaris. ARVO Abstracts. Invest Ophthalmol Vis Sci 42:S437, 2001

40.Pratt LM, Criswell MH, Ciulla TA, Danis RP, Hill TE, Snyder WJ, Small W: Choriocapillaris closure in normal primates using photosensitizer MV6401 at different postinjection activation times. ARVO Abstracts. Invest Ophthalmol Vis Sci 42:S437, 2001

41.Framme C: Personal communication

42.Mori M, Kuroda T, Obana A, Sakata I, Hirano T, Nakajima S, Hikida M, Kumagai T: In vitro plasma protein binding and cellular uptake of ATX-S10(Na), a hydrophillic chlorine photosensitizer. Jpn J Cancer Res 91:845-852, 2000

43.Schmidt-Erfurth U, Hasan T, Gragoudas E, Michaud NA, Flotte TJ, Birngruber R: Vascular targeting in photodynamic occlusion of subretinal vessels. Ophthalmology 101(12): 1953-1961, 1994

44.Vlodavsky I, Fielding PE, Fielding CJ, Gospodarowicz D: Role of contact inhibition in the regulation of receptormediated uptake of low-density lipoprotein in cultured vascular endothelial cells. Proc Natl Acad Sci USA 75:356360, 1978