December 2006
Volume 47, Issue 12
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Retina  |   December 2006
Evaluation of the New Photosensitizer Tookad (WST09) for Photodynamic Vessel Occlusion of the Choroidal Tissue in Rabbits
Author Affiliations
  • Carsten Framme
    From the Medical Laser Center Lübeck, Lübeck, Germany; and the
    University Eye Hospital Regensburg, Regensburg, Germany.
  • Helmut G. Sachs
    University Eye Hospital Regensburg, Regensburg, Germany.
  • Barbara Flucke
    From the Medical Laser Center Lübeck, Lübeck, Germany; and the
  • Dirk Theisen-Kunde
    From the Medical Laser Center Lübeck, Lübeck, Germany; and the
  • Reginald Birngruber
    From the Medical Laser Center Lübeck, Lübeck, Germany; and the
Investigative Ophthalmology & Visual Science December 2006, Vol.47, 5437-5446. doi:10.1167/iovs.06-0532
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      Carsten Framme, Helmut G. Sachs, Barbara Flucke, Dirk Theisen-Kunde, Reginald Birngruber; Evaluation of the New Photosensitizer Tookad (WST09) for Photodynamic Vessel Occlusion of the Choroidal Tissue in Rabbits. Invest. Ophthalmol. Vis. Sci. 2006;47(12):5437-5446. doi: 10.1167/iovs.06-0532.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the efficacy of Tookad (WST09; Negma-Lerads, Magny-Les-Hameaux, France) photodynamic therapy (T-PDT) by evaluating the angiographic and histologic closure of choroidal vessels at different radiance exposures, drug dosages, and intervals between photosensitizer injection and laser application in a rabbit model.

methods. Chinchilla Bastard rabbits were injected intravenously with three different dye concentrations (2.5, 5, and 10 mg/kg) before application of light. In every group T-PDT was performed at four different times after injection: 5, 15, 30, and 60 minutes with different radiance exposures ranging from 200 to 3 J/cm2. Fundus photographs and fluorescein angiograms were obtained 90 minutes after injection. Follow-up angiographies were performed at days 1, 3, 7, and 14 after initial treatment. Histology was performed in selected cases immediately after treatment and on days 1, 3, and 7.

results. Immediately after irradiation, most of the visible lesions were angiographically hyperfluorescent due to damaged vessel endothelium and associated RPE damage. Lesions from high-radiance exposures revealed immediate hypofluorescence, indicating vessel closure. Hypofluorescent lesions appeared mainly during day 1 (all lesions angiographically visible, some hypofluorescent) to day 3 (all lesions hypofluorescent) after treatment. At day 7, ophthalmoscopically visible hyperpigmentation took place in all lesions. ED50thresholds for angiographic hypofluorescence determined at day 3 after treatment with 2.5 mg/kg were 18.8 J/cm2 (5 minutes), 62.0 J/cm2 (15 minutes), and >100 J/cm2 (30 minutes); with 5 mg/kg, 8.4 J/cm2 (5 minutes), 22.8 J/cm2 (15 minutes), 54.5 J/cm2 (30 minutes), and >100 J/cm2 (60 minutes); and with 10 mg/kg, 11.7 J/cm2 (30 minutes) and 54.1 J/cm2 (60 minutes). Histology of the angiographically hypofluorescent lesions revealed vessel thrombosis in all groups 1 hour after PDT up to 7 days after treatment. Sparing of photoreceptors indicated selectivity of T-PDT; however, slight damage was partly observable. After 7 days, localized proliferation of the RPE cells was noted and was enhanced 14 days after treatment.

conclusions. T-PDT has the potential to achieve selective choroidal vessel occlusion with proper parameter selection, such as (1) 2.5 mg/kg, 5 minutes, 100 J/cm2; (2) 5 mg/kg, 5 minutes, 25 J/cm2; or (3) 5 mg/kg, 15 minutes, 50 J/cm2; however, slight damage to the photoreceptors cannot be ruled out. RPE proliferation indicates primary RPE damage due to PDT, also described with the use of all other photosensitizers.

Photodynamic therapy (PDT) involves intravenous injection of a photosensitizer that accumulates in neovascular and tumor tissue. Βy irradiating the photosensitized tissue with light at the absorption maximum of the dye, cytotoxicity can be achieved. 1 2 For neovascular age-related macular degeneration (AMD), which is the leading cause of blindness in patients older than 65 years in the industrialized nations, 3 4 5 6 PDT using verteporfin has been widely thought during the past years to be successful in preventing visual loss. 7 Current approved PDT patterns for treatment of neovascular AMD involves the injection of benzoporphyrin derivatives (BPDs; verteporfin) and irradiation with 689 nm for 83 seconds 15 minutes after injection (600 mW/cm2; 50 J/cm2), which shows the best clinical results for predominantly classic lesions. 7 Currently, numerous second-generation photosensitizers have been tested for treatment of neovascular AMD 8 9 10 11 ; however, some disadvantages of each of these photosensitizers remain. 
The photosensitizer used in this study was bacteriochlorophyll (Bchl; Tookad, [WST09]; Negma-Lerads, Magny-Les-Hameaux, France), a lipophilic, water-soluble derivative of the natural pigment Bchl with an incorporated palladium (Pd) atom and a maximum excitation peak of 762 nm (high-absorption coefficient of 105); 12 13 14 15 16 ; (Fig. 1) . Further characteristics of the dye are molecular weight of 714, purity > 95%, stability in air, low rate of photobleaching, and additional spectral peaks at 530, 385, and 330 nm. After intravenous administration, Tookad becomes mainly bound to HDL and LDL proteins. It has an extremely strong vascular effect. The quantum efficiency for triplet state production is approximately 99%, with very high production of singlet oxygen, the putative toxic agent in PDT (type II mechanism). Further studies on the photochemistry of Tookad have revealed the highest photodynamic effect shortly after injection, 15 which seems to be due to ultrafast electron transfer from one Bchl to another in loosely coupled dimmers. The anion can reduce the oxygen presence, starting a chain of events ending with the ejection of the hydroxyl radical, an anion in a non-Fenton reaction. 15 Because the radicals seem to be ejected directly from the excited sensitizer, it is suspected that their formation falls into neither a type I nor II process but represents a class by itself: a type III process. 15  
Clinically, the main advantages of Tookad in comparison with other tested sensitizers are as follows: (1) Because of its high-wavelength absorbance, the exciting light beam is able to penetrate deeper into tissues and thus may permit an enhanced selective treatment of the choroidal vessels behind the strong absorbing RPE. (2) Because of its strong optical absorption, excitation with low-energy light sources is possible. (3) A short delay between injection and irradiation causes mainly vascular damage. (4) The action of Tookad is rapid (phototoxicity >200 times stronger than with Photofrin II; Axcan, Mont Saint Hilaire, Québec, Canada), with treatment being completed within 1 hour, and it clears rapidly from the blood circulation (<24 hours in mice 14 ). This feature permits ambulatory treatment and presents a low risk of adverse complications such as photosensitizing of the skin after treatment. (5) Animal experiments show no drug toxicity (in the dark) at a dose 100 times higher than the effective treatment dose. 14  
The purpose of this study was to determine the ability of Tookad to produce choroidal vascular occlusion when different treatment parameters were used in a rabbit model. 
Material and Methods
Lasers
PDT lesions were performed with an argon, pumped-wavelength tuneable ti:sapphire laser. The wavelength was continuous wave (cw) 762.0 ± 0.1 nm (full width at half maximum [FWHM] 0.2 nm). The laser beam was delivered to a clinically used ophthalmic laser slit lamp (model 166788; Carl Zeiss Meditec, GmbH, Oberkochen, Germany) using a multimode fiber (160 μm, 0.1 NA). The magnification of coupling was sixfold. The laser beam was focused onto the retinal surface in the central area of the rabbit eye by a contact lens (Goldmann 903; three mirror; Haag Streit, Köniz, Switzerland). With the use of a magnification factor of 0.66 when irradiating rabbit’s eyes with a plano-concave contact lens in cycloplegic emmetropic eyes, 17 the retinal spot size appeared to be 634 μm. Irradiation was performed with 600 mW/cm2 and an intensity of 1.89 mW ± 10%. The aiming laser emitted at 635 nm. To avoid activation of the photosensitizer due to illumination, we integrated a blue filter (BP459) into the slit lamp. 
Ophthalmoscopically visible marker lesions are necessary for the invisible PDT lesions, to demarcate the treatment area and were created by the use of an arc-lamp–excited, intracavity, frequency-doubled Nd:YLF laser (model 527DP-H; Quatronix, Inc., Akron, OH), emitting at a wavelength of 527 nm. 18 Suprathreshold irradiation with pulse durations of 1.7 μs (30 pulses, 100 Hz) were achieved. The energy was transmitted by a 105-μm core diameter fiber (Optran UV-A 105/125/250, NA 0.1; Ceram Optec GmbH, Bonn, Germany), which was directly coupled to the slit lamp fiber (diameter, 158 μm; NA 0.1; Carl Zeiss Meditec, Inc.). 
Photosensitizer
Tookad (bacteriochlorophyll; Bchl) is a water-soluble derivative of the natural pigment Bchl with an incorporated palladium atom. The preparation has been described by Scherz et al. 19 The spectrum of Tookad is displayed in Figure 1 , showing four maximum peaks at 330, 385, 530, and 762 nm, which is the predominant fluorescent peak used for excitation. The preparation of Tookad used in this study had a pH of 7.56 and an osmolarity of 813. 
Animals
Chinchilla Bastard rabbits were used for the experiments. Each animal weighed between 2.0 and 3.0 kg. Rabbits were chosen because the density and location of light-absorbing pigments in the fundus are rather uniform and similar to that in the human eye. 20 The animals were anesthetized with ketamine hydrochloride (35 mg/kg of body weight) and xylazine hydrochloride (5 mg/kg of body weight). Pupillary dilatation was achieved with topical application of phenylephrine hydrochloride 2.5% and tropicamide 1%. The animals were placed in a special holder system that allowed movement in all directions. The plano-concave contact lens was placed on the mydriatic eye with methylcellulose used as a contact gel. The lens was locked in the animal holder to prevent movement. After administration of the dye, the animals were kept in dark conditions for 3 days. Usually they were housed in rooms with ordinary fluorescent lamps (12 hours on, 12 hours off). Enucleations were performed with the animals under deep anesthesia, after which they were euthanatized by an intravenous injection of pentobarbital sodium. 
The treatment of experimental animals in this study was in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Laser Treatment and Documentation
The particular nonvisible PDT lesions (n = 16 per eye for threshold evaluation) were placed between the marker lesions (n = 6 marking a field of approximately 6 × 6 mm in the central retina) enabling orientation during treatment. For the threshold experiments, 24 eyes were irradiated. Animals were separated into three groups with Tookad concentrations of 2.5 mg/kg (group 1), 5 mg/kg (group 2), and 10 mg/kg (group 3). In the first 15 eyes (5 eyes per group), radiance exposures of 100, 50, 25, and 12 J/cm2 were applied at each of 5, 15, 30, and 60 minutes after dye injection. A typical irradiation pattern is shown in Fig. 2 . In another nine eyes, irradiation patterns were slightly changed including 200, 6, and 3 J/cm2 for the preliminary calculated thresholds to gain more lesions at the supposed vessel occlusion threshold. Thus, for each of the single parameters (n = 48) at least 5 and at most 14 lesions could be evaluated. 
Ninety minutes after application of the laser lesions, fundus photographs were taken with a fundus camera (Carl Zeiss Meditec GmbH). Afterward, standard fluorescein angiography was performed, with injection of 10% fluorescein sodium (2 mL bolus) into the ear vein. 
For evaluation of presumed vessel occlusion, the angiographic hypofluorescence in each PDT lesion was plotted versus the laser energy for the various fluences. From the plot, the mean threshold radiance exposures for the different fluences were determined. The angiographic threshold irradiation (ED50) was defined as the irradiation necessary to achieve fluorescein angiographically hypofluorescent vessel closure with a 50% probability. For presentations of the ED50 the y-axis was plotted as an inverse normal distribution function (probability plot) versus the logarithmically calculated energy, resulting in a line of y = ax. 21 22 The thresholds were calculated using software for probit analysis. 23 It should be noted that angiographic hypofluorescence may only signal possible vessel occlusion, which can be verified only by subsequent histology. Also, edema may block choroidal fluorescence leading to hypofluorescence. However, hypofluorescence was often seen without edema and was additionally proven by histology; thus, hypofluorescence could be regarded as a strong indicator for choroidal vessel occlusion. 
Histology
Based on the obtained threshold results, selected laser parameters in groups 1 (2.5 mg/kg) and 2 (5 mg/kg) were chosen to be evaluated histologically. For this purpose, suprathreshold lesions were placed in a suitable irradiation pattern consisting of 2 × 3 lesions per parameter per eye (n = 12; Fig. 3 ). Fluorescein angiography was performed at day 1 after treatment to verify vascular thrombosis for each lesion, as judged by angiographic hypofluorescence. Histology was performed at 1 hour, and 1, 3, and 7 days after irradiation. Immediately after enucleation, the globes were incised anterior to the equator and immersed in 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer. The posterior eye cup was cut from the anterior segment after 10 minutes and replaced in the fixative. The retinas were then fixed in 2.5% glutaraldehyde and postfixed in Dalton’s osmium fixative, dehydrated in alcohol, and embedded in epoxy resin (Epon). Ultrathin sections were stained with uranyl acetate. One-micrometer serial sections were cut until the center of the lesion was reached. The status of occlusion of the choriocapillaries and the choroid was investigated, as well as the collateral damage of the RPE and the photoreceptors. 
Results
Angiographic Evaluation
Angiography was performed at 1 hour, and 1, 3, 7, and 14 days after irradiation. Usually, at 1 hour most of the lesions that were detectable by angiography revealed hyperfluorescent leakage; however, at high radiance exposures (100 J/cm2) initial hypofluorescence indicating vessel occlusion was also detected. After 1 day, more lesions were detectable (previously ophthalmoscopically invisible lesions became visible after 1 day), and most of them were hypofluorescent, but some hyperfluorescent lesions at lower radiance exposures were also observed. At day 3, all detectable lesions were hypofluorescent. Hypofluorescence was stable up to day 7; however, some changes in angiographic patterns were noted that were caused by ophthalmoscopically visible hyperpigmentation, which blocked choroidal fluorescence in part. The same conditions were found 14 days after treatment, with enhanced hyperpigmentation of the fundus within the lesion area. Some reperfusion at that time cannot be ruled out (Fig. 4) . Fewer lesions were found in the 5-mg/kg group than in the 10-mg/kg group and subsequently in the 2.5-mg/kg group; however, the described patterns were the same. 
Ophthalmoscopic Evaluation
According to the angiographic results, most lesions were not visible 1 hour after irradiation and became whitish at day 1. On day 3 after treatment, the whitish color of the lesions enhanced but regressed again at day 7 (Fig. 4) . In all angiographically detectable lesions—independent of drug dosage, radiance exposure, and time interval between injection and irradiation—a hyperpigmentation was observed at day 7 after treatment. Thus, for no particular laser parameter, choroidal vessel occlusion was seen that had no effect on the RPE. However, it was noted that hyperpigmentation was enhanced when radiance exposure was highly suprathreshold (Fig. 4)
Efficacy of Tookad Regarding Different Time Intervals between Injection and Irradiation
Table 1shows the efficacy of Tookad in terms of irradiation at different times after injection as determined at day 3 after treatment (5 mg/kg dye concentration), when PDT effects resulted in maximum angiographic hypofluorescence. Irradiation took place in all eyes 5, 15, 30, and 60 minutes after injection. It was clearly demonstrated that, as the time interval between the Tookad injection and irradiation was lengthened, the rate of detectable hypofluorescent lesions decreased significantly also in accordance with the radiance exposures used (Table 1)
Threshold Power Levels for Achieving Angiographically Hypofluorescent Lesions
Figure 5shows the calculation of the ED50 threshold power level for the 5-mg/kg dye concentration when irradiation took place 15 minutes after injection. Hypofluorescent lesions were achieved at an ED50 threshold of 22.8 J/cm2 (Fig. 5) . Table 2summarizes Tookad’s efficacy at various dosages and radiance exposures, as determined by fluorescein angiography at day 3 after treatment. In group 1 (2.5 mg/kg), vascular occlusion was achieved at calculated ED50 thresholds of 18.8 J/cm2, when irradiation took place 5 minutes after dye injection. Irradiation 15 minutes after injection led to a higher ED50 threshold at 62.0 J/cm2, and 30 minutes after injection, the threshold was higher than 100 J/cm2. No treatment effect was observable with irradiation 60 minutes after injection. In group 2 (5 mg/kg), thresholds usually were significantly lower, with an ED50 of 8.4 J/cm2 (5 minutes), 22.8 J/cm2 (15 minutes), 54.5 J/cm2 (30 minutes), and >100 J/cm2 (60 minutes). In group 3 (10 mg/kg), irradiation 5 and 15 minutes after dye injection led to vascular occlusion in all lesions at all radiance exposures. The ED50 threshold for the 30-minute time interval was 11.7 J/cm2, and for 60 minutes it was 54.1 J/cm2 (Table 2)
Histology
Based on the ED50 results for feasible clinical treatments, histology was performed for treatment with 2.5 and 5 mg/kg Tookad. Twelve specimens were examined histologically according to the parameters in Table 3 . For treatment with 5 mg/kg dye concentration, histology was evaluated 1 hour and 7 days after treatment (irradiation 15 minutes after injection with 50 J/cm2 and 30 minutes after injection with 100 J/cm2). For both irradiation patterns, 1 hour after treatment the lesions were ophthalmoscopically nonvisible, and angiography revealed focal hypofluorescence. In these areas, histology showed undamaged photoreceptors, a thinner RPE layer, and only some focal occlusion of the choriocapillaris and choroidal vessels (Fig. 6) . For specimens with the same parameters, 7 days after treatment, angiography showed hypofluorescence and histology revealed occluded vessels and intact photoreceptors (Fig. 7) . Most striking is the finding of proliferating RPE with extensive nodular proliferations leading to multilayered RPE (Figs. 8 9) . Proliferation was more intense when irradiation at 100 J/cm2 was applied 30 minutes after dye injection. 
Regarding 2.5 mg/kg Tookad, irradiation at 5 and 15 minutes after dye application (each 100 J/cm2) was examined. One hour after treatment, both parameters revealed histologically intact photoreceptors and open vessels within the choriocapillaris and choroid but a thinner RPE layer (Fig. 10) . The lesions were ophthalmoscopically invisible, and angiography did not show any hypofluorescence. One day later, lesions were ophthalmoscopically visible as light spots and were angiographically hypofluorescent. Despite these findings, histology showed partly occluded vessels but still some open vessels. The photoreceptor layer was intact, but RPE was dense, thin, and hyperpigmented (Fig. 11) . Three days after treatment, the vessels were occluded, slight damage to the photoreceptors was observed, and some proliferation of the RPE was noted (Fig. 12) . Histology 7 days after treatment revealed occluded vessels for both parameters, RPE proliferation, and slight damage to the photoreceptors (Fig. 13) . No nodular RPE proliferation, as was seen for the 5-mg/kg dye injection, was noted at the lower dye concentration. 
Discussion
Today, just one photosensitizer, verteporfin, is approved for use in clinical treatment of neovascular AMD. However, there are several photosensitizers currently being tested in experimental and clinical studies. 8 9 10 11 Tookad has some advantages in contrast to other sensitizers. One is strong absorption at 770 to 780 nm in the infrared-A range, which enables greater tissue penetration. In addition, Tookad overcomes many of the disadvantages especially of the hematoporphyrin derivatives, because it has been shown to be approximately 200 times more phototoxic and to clear much more rapidly from tissues. 14 24  
The purpose of this preclinical study was to evaluate Tookad’s potential for occlusion of the choriocapillary vessels in the eye. Angiographic evaluation was performed at five different times throughout the study. It was observed that early changes as determined 1 hour and 1 day after treatment are not sufficient to indicate final vessel occlusion, because more lesions (hypofluorescent and hyperfluorescent ones) were detectable at 1 day than at 1 hour after irradiation, indicating a time-dependent biological process induced by PDT. This thrombotic process was fast for high-radiance exposures, as seen for 100 J/cm2, but it may have taken at least 1 to 3 days for lower radiance exposures slightly above threshold. Thus, complete vessel occlusion derived from angiographically seen hypofluorescence could not be finally judged earlier than day 3. Moreover, the vessel endothelium was always damaged, and as shown by histology, the RPE was also damaged, leading to leakage and pooling of the fluorescein in the subretinal space, effectuating an angiographic hyperfluorescence. This effect raised difficulties in properly judging hypofluorescent lesions, especially within the first 2 days after treatment, when pooling was very fast and angiography was performed using static images of the camera (Carl Zeiss Meditec). 
Ophthalmoscopically, the PDT lesions appeared whitish, presumably due to retinal edema in the first 3 days, more enhanced at day 3 than at day 1. At day 7, the lesions faded, and hyperpigmentation was even more pronounced at day 14. With irradiation much above threshold, as seen for 10 mg/kg Tookad with radiance exposures of 100 J/cm2 5 minutes after injection, occasional distinctive fibrosis was noted in the irradiated area. Because of the hyperpigmentation, a blocking phenomenon was present that suggested angiographic hypofluorescence in part (Figs. 4L 4M) . Thus, possible reperfusion of the capillaries could not be determined exactly in all lesions at this time. Extensive hyperpigmentation, as described herein, is often seen in laser trials in the rabbit retina, because these animals are known to have a predisposition for strong RPE proliferation. It is assumed that no gross proliferation takes place in humans; however, a distinct impact of RPE damage from Tookad (T)-PDT cannot be ruled out in humans, as has also been true of all other photosensitizers. 
ED50 thresholds were lowest with irradiation at 5 minutes after dye injection for 10 mg/kg (< 8 J/cm2) followed by 5 mg/kg (8.4 J/cm2) and 2.5 mg/kg (18.8 J/cm2) Tookad. With the 10-mg/kg dosage, significant ED50 levels at tested radiance exposures were obtained primarily after 30 minutes (at 5 and 15 minutes all lesions were positive for vessel occlusion), which means a very high concentration of dye and a long waiting time between dye application and irradiation to avoid overtreatment—problems that may make this dye concentration inadvisable for clinical use. Thus, histologic examination of these parameters was not routinely performed. Regarding 2.5 mg/kg, clinically reasonable results were achieved with irradiation at 5 and 15 minutes after treatment; however, 100% probability of generating vessel occlusion was seen only at 100 J/cm2 (5 minutes after injection) which resulted in an irradiation time of 164 seconds, relatively long for clinical use. Finally, a dye concentration of 5 mg/kg seems to be appropriate for Tookad. Reasonable irradiation parameters might be 25 J/cm2 5 minutes after injection or 50 J/cm2 at 15 minutes. If these proposed parameters are used, the probability of achieving angiographic vessel occlusion is approximately 100% and thus, irradiation is already considered to be significantly higher than is needed for the ED50 threshold. 
Comparing all calculated thresholds, as derived from Figure 5and Table 2 , it is notable that with irradiation with a double dose of dye or an approximately doubled time interval after dye injection, the threshold increase was always approximately two to three times (except for the 10 mg/kg, 30-minute parameter), which is reasonable and shows that the threshold values were correctly determined (Fig. 14) . With the proposed parameters, time intervals between dye application and final laser treatment were short—comparable to the current PDT treatments with verteporfin—which may be a benefit for the patient, because it eliminates waiting time before PDT. These data also showed that the efficacy of Tookad 60 minutes after injection was low in all groups. This finding indicates the fast clearance time of the dye from the blood circulation, which lowers the chance of side effects such as skin burns, which were often seen during the TAP investigations with verteporfin. 7  
Histology revealed the potential of Tookad to occlude the choriocapillaris and the choroidal vessels at 2.5- and 5-mg/kg dye concentrations, clearly seen 3 to 7 days after treatment. The observed delay in achieving vessel occlusion after treatment supports the conclusion that vessel occlusion on a photodynamic basis is a dynamic process that predominantly takes place between the first and third days after treatment, if irradiation is slightly suprathreshold. Different mechanisms for the vascular occlusion due to PDT were discussed; however, the main reason is thought to be a huge release of factor VIII or thromboxin after PDT from the damaged endothelial cells, leading to aggregation of thrombocytes. 25 Because of this—especially at threshold irradiation—a primary apposition thrombosis may occur at the vessel intima, leading to turbulence in the blood flow and consecutive apposition of material at this site and finally to occlusion of the complete vessel lumen. This process is known to be dynamic, depending on dye concentration, radiance exposure, and time of irradiation. Thus, in our investigation, histology revealed partly open vessels 1 day after treatment, despite successful irradiation, but closure of all vessels 3 days after irradiation (Table 3) . Presumed partial reperfusion of the lesions as assumed from the angiographic findings due to blockage by hyperpigmentation could not be confirmed by the histologic examinations; thus, Tookad seems to have a robust potential for achieving proper vessel occlusion. 
The problem of RPE damage due to PDT is very well known and is also described with verteporfin treatment. 26 27 In our study, the RPE was found to be thinner than normal RPE within the first 3 days after treatment and was partly dense and more highly pigmented. After 7 days at both concentrations, RPE proliferation was histologically obvious, clearly demonstrating damage to the RPE. Some studies suggest that intact RPE cell layers cover the site of prior photodynamic injury within a few weeks. 17 28 However, it is also known that rabbit RPE is highly reactive in contrast to human RPE; thus, the extensive proliferation as partly seen in this study may not occur in humans. Proliferation has to be regarded as a healing process for damaged RPE and is widely described with all other photosensitizers that are currently under investigation. This finding is in fact very important because it may reduce the selectivity of this method significantly. 
Besides verteporfin, the other major photosensitizers of the second generation, which have entered preclinical and clinical trials, are SnET2 (tin ethyl etiopurpurin; purlitin), lutetium texaphyrin (Lutex; Alcon, Fort Worth, TX), mono-l-aspartyl chlorine e6 (NPe6), and ATX-S10. 
Purlitin, a lipophilic sensitizer, photoactivates at 664 nm and occludes choriocapillary vessels successfully in pigmented rabbits when irradiation starts 15 to 45 minutes after dye injection at an irradiance of 300 mW/cm2 and relatively light doses of 5 to 20 J/cm2. RPE damage and outer retinal alterations were documented using the optimal parameters for vessel occlusion (Moshfegi DM, et al. IOVS 1995;36:ARVO Abstract 115). 10 This photosensitizer has already been evaluated in clinical trials with vision results comparable to those of verteporphin therapy; however, treated patients have to avoid bright light for several weeks because of prolonged retention of the sensitizer within the skin. 29 Moreover, in comparison to Tookad, water solubility is poor, and the extinction coefficient is only a third of Tookad’s. 30  
Lutetium texaphyrin, a water-soluble, synthetic porphyrin analogue, photoactivates at 732 nm, improving tissue transmission due to the longer wavelength. 31 In an experimental model of laser-induced choroidal neovascularization (CNV) in the monkey, absence of fluorescein leakage from the CNV lesion was obtained with treatment using 2 mg/kg sensitizer and 50 or 100 J/cm2 at an irradiance of 600 mW/cm2(Arbour JD, et al. IOVS 1999;40:ARVO Abstract 401). 29 Occlusion of the choriocapillary layer was found in all parameters tested; however, damage to the neurosensory layer and necrosis of the RPE was also described (Arbour JD, et al. IOVS 1999;40:ARVO Abstract 401). A clinical trial is under way. Because the extinction coefficient is also only a third of Tookad’s, 30 the photodynamic effect is regarded to be less. Human plasma half-lives are brief (0.25–8.8 hours), which is a considerable advantage. 30  
NPe6, a hydrophilic component, photoactivates at 664 nm and was evaluated preclinically in rabbits and monkeys. At concentrations of 2 mg/kg, occlusion of the choriocapillary layer was achieved at above 2.65 J/cm2 in pigmented rabbits and above 0.88 J/cm2 in nonpigmented animals. 32 The described side effects were retinal thinning, loss of photoreceptor outer segments, and RPE proliferation. 32 33 Long human plasma half-lives of 9 to 134 hours are disadvantageous and also the extinction coefficient is considerably lower than that of Tookad. 30  
Another water-soluble photosensitizer in animal studies is ATX-S10, which has selectively occluded experimental CNV in a rat model (Obana A, et al. IOVS 1998;39:ARVO Abstract 389). ATX-S10 photoactivates at 670 nm, and vascular occlusion has been evaluated at various drug doses, irradiances, radiance exposures, and drug–light intervals in rats and monkeys. 34 35 Although long-term occlusion was achieved up to 28 days, RPE damage was observed and described to recover in rats. 34 However, in monkeys RPE proliferation with pigment-laden cells and double-layered RPE has been reported. 35 The clearance rate is rapid (in rabbits, 45 minutes); however, the extinction coefficient is very low. 30  
Because animal models and parameters used in different photosensitizer studies are heterogeneous, the extent of RPE damage cannot be compared; however, the RPE reaction of Tookad is expected to be significantly lower in human trials. Compared with other photosensitizers, the main advantage of Tookad seems to be photoactivation at the highest wavelength of all sensitizers with the best passage into the deeper tissues of the choriocapillaris. Moreover, it has a rapid clearance, leading to fewer side effects such as skin burns, and exhibits the largest extinction coefficient at a factor of 105. However, it is considerably debatable whether higher-wavelength activation really leads to a more selective effect or whether unwanted side effects such as choroidal occlusion are enhanced. Even when longer wavelengths are used, the extremely light-sensitive RPE will be harmed, then producing cytotoxic free radicals released into the cytosol and consecutively damaging the RPE. 30 The recurrence rate of choroidal neovascularization in clinical application using BPD is approximately 50%. 36 Because the cellular phototoxicity of Tookad is regarded to be higher than that of other sensitizers, it is supposed that the recurrence rate may be decreased in neovascular AMD treated with Tookad. 
In summary, T-PDT was first evaluated for ophthalmologic concerns in this preclinical study for dose-range determination. It was shown that T-PDT has the potential to achieve choroidal vessel closure. Proper irradiation parameters may favor 5 mg/kg dye concentration and irradiation 5 minutes after dye application at a radiance exposure of 25 J/cm2 or 15 minutes after dye application at a radiance exposure of 50 J/cm2. Unwanted side effects such as RPE proliferation were observed but have also been described for all other photosensitizers tested so far. Also, slight photoreceptor damage could not be ruled out in all cases. However, due to the high wavelength and deeper tissue penetration of laser light, collateral damage to the neurosensory layer may be less in humans than with other photosensitizers. Thus, human studies are necessary to determine the future role of this agent in the treatment of AMD. 
 
Figure 1.
 
Chemical appearance and excitation spectrum of Tookad, which is a water soluble derivative of the natural pigment bacteriochlorophyll. A Palladium atom is incorporated within the central part of the molecule. The peak with the highest absorption coefficient is at 762 nm. There are additional peaks at 530, 385, and 330 nm.
Figure 1.
 
Chemical appearance and excitation spectrum of Tookad, which is a water soluble derivative of the natural pigment bacteriochlorophyll. A Palladium atom is incorporated within the central part of the molecule. The peak with the highest absorption coefficient is at 762 nm. There are additional peaks at 530, 385, and 330 nm.
Figure 2.
 
Irradiation pattern for the application of the 16 PDT lesions per rabbit eye. The X stands for the marker lesions. The first row of PDT lesions was applied 5 minutes after dye injection, the third row at 15 minutes, the second row at 30 minutes, and the fourth row at 60 minutes. Irradiation took place from 100 to 12 J/cm2 initially.
Figure 2.
 
Irradiation pattern for the application of the 16 PDT lesions per rabbit eye. The X stands for the marker lesions. The first row of PDT lesions was applied 5 minutes after dye injection, the third row at 15 minutes, the second row at 30 minutes, and the fourth row at 60 minutes. Irradiation took place from 100 to 12 J/cm2 initially.
Figure 3.
 
(a) Typical irradiation pattern of PDT lesions assigned to be examined histologically (X, marker lesion; A and B, lesions with the same irradiation parameters); (b) Photograph of lesions 1 day after irradiation.
Figure 3.
 
(a) Typical irradiation pattern of PDT lesions assigned to be examined histologically (X, marker lesion; A and B, lesions with the same irradiation parameters); (b) Photograph of lesions 1 day after irradiation.
Figure 4.
 
Early angiography (a), late angiography (b), and fundus photograph (c) 1 hour after irradiation (10 mg/kg Tookad dye injection). The early-phase angiography revealed hypofluorescence, suggesting choroidal vessel occlusion in three lesions (5 minutes: 100 and 50 J/cm2; 15 minutes: 100 J/cm2). Late-phase angiography revealed additional damage by leakage in three other lesions. In the fundus photograph a slight whitening of two lesions is visible. Early angiography (d), late angiography (e), and fundus photograph (f) 1 day after irradiation in the same eye. Early and late angiography phases revealed hypofluorescence suggesting choroidal vessel occlusion in the first three lesions of the first three rows. Additional leakage was found for the 12-J/cm2 lesions in the first three rows and additionally for 100 and 50 J/cm2 in the fourth row. Also notable was that the fast pooling effect in the irradiated area enhanced whitening with all but one parameter (12 J/cm2, 60 minutes after dye injection). Early angiography (g), late angiography (h), and fundus photograph (i) 3 days after irradiation in the same eye. Early-phase angiography revealed hypofluorescence suggesting choroidal vessel occlusion in all but two lesions (25 and 12 J/cm2, 60 minutes after dye injection). Fast pooling was still present, as seen in the late-phase angiography. Fundus photograph shows light lesions, with the especially the first two lesions in the first three rows being very pronounced. Early angiography (j) and fundus photograph (k) 7 days after irradiation in the same eye. The fundus photograph reveals hyperpigmentation in the irradiated areas and fading of the whitening. In some areas, a sort of fibrosis occurred. Because of the hyperpigmentation, a blockade of the fluorescein in the angiography appeared, thus leading to hypofluorescence exactly corresponding with the pigmentation. Early angiography (l) and fundus photograph (m) 14 days after irradiation in the same eye. A fundus photograph revealed enhanced hyperpigmentation over the whole area, with corresponding angiographic hypofluorescence.
Figure 4.
 
Early angiography (a), late angiography (b), and fundus photograph (c) 1 hour after irradiation (10 mg/kg Tookad dye injection). The early-phase angiography revealed hypofluorescence, suggesting choroidal vessel occlusion in three lesions (5 minutes: 100 and 50 J/cm2; 15 minutes: 100 J/cm2). Late-phase angiography revealed additional damage by leakage in three other lesions. In the fundus photograph a slight whitening of two lesions is visible. Early angiography (d), late angiography (e), and fundus photograph (f) 1 day after irradiation in the same eye. Early and late angiography phases revealed hypofluorescence suggesting choroidal vessel occlusion in the first three lesions of the first three rows. Additional leakage was found for the 12-J/cm2 lesions in the first three rows and additionally for 100 and 50 J/cm2 in the fourth row. Also notable was that the fast pooling effect in the irradiated area enhanced whitening with all but one parameter (12 J/cm2, 60 minutes after dye injection). Early angiography (g), late angiography (h), and fundus photograph (i) 3 days after irradiation in the same eye. Early-phase angiography revealed hypofluorescence suggesting choroidal vessel occlusion in all but two lesions (25 and 12 J/cm2, 60 minutes after dye injection). Fast pooling was still present, as seen in the late-phase angiography. Fundus photograph shows light lesions, with the especially the first two lesions in the first three rows being very pronounced. Early angiography (j) and fundus photograph (k) 7 days after irradiation in the same eye. The fundus photograph reveals hyperpigmentation in the irradiated areas and fading of the whitening. In some areas, a sort of fibrosis occurred. Because of the hyperpigmentation, a blockade of the fluorescein in the angiography appeared, thus leading to hypofluorescence exactly corresponding with the pigmentation. Early angiography (l) and fundus photograph (m) 14 days after irradiation in the same eye. A fundus photograph revealed enhanced hyperpigmentation over the whole area, with corresponding angiographic hypofluorescence.
Table 1.
 
Efficacy of Tookad at Different Time Intervals between Injection and Irradiation in Rabbits That Had Received 5 mg/kg
Table 1.
 
Efficacy of Tookad at Different Time Intervals between Injection and Irradiation in Rabbits That Had Received 5 mg/kg
5 Minutes 15 Minutes 30 Minutes 60 Minutes
100J: 5/5 100% 100J: 5/5 100% 100J: 6/8 75% 100J: 1/5 20%
50J: 8/8 100% 50J: 6/7 86% 50J: 4/10 40% 50J: 0/5 0%
25J: 12/12 100% 25J: 5/10 50% 25J: 2/7 29% 25J: 0/5 0%
12J: 8/11 73% 12J: 2/8 25% 12J: 0/5 0% 12J: 0/5 0%
Figure 5.
 
Probability of choroidal vessel occlusion versus the applied radiance exposure for a drug dose of 5 mg/kg. Irradiation took place 15 minutes after injection of the dye. The ED50 threshold for this parameter was 22.8 J/cm2.
Figure 5.
 
Probability of choroidal vessel occlusion versus the applied radiance exposure for a drug dose of 5 mg/kg. Irradiation took place 15 minutes after injection of the dye. The ED50 threshold for this parameter was 22.8 J/cm2.
Table 2.
 
Threshold Power Levels Needed for the Laser Parameters Used to Achieve Angiographically Determined Vessel Occlusion of the Choriocapillaris as Evaluated at Day 3 After Treatment
Table 2.
 
Threshold Power Levels Needed for the Laser Parameters Used to Achieve Angiographically Determined Vessel Occlusion of the Choriocapillaris as Evaluated at Day 3 After Treatment
2.5 mg/kg 5 mg/kg 10 mg/kg
5: 18.8 J/cm2 5: 8.4 J/cm2 5: <8.4 J/cm2
15: 62.0 J/cm2 15: 22.8 J/cm2 15: <8.4 J/cm2
30: >100.0 J/cm2 30: 54.5 J/cm2 30: 11.7 J/cm2
60: — 60: >100.0 J/cm2 60: 54.1 J/cm2
Table 3.
 
Summary of Histologically Examined Laser Lesions Induced by the Different Laser Parameters That Are Capable of Leading to Vessel Occlusion of the Choriocapillaris
Table 3.
 
Summary of Histologically Examined Laser Lesions Induced by the Different Laser Parameters That Are Capable of Leading to Vessel Occlusion of the Choriocapillaris
Drug Dose (mg) Days Post. Treat Parameter Ang. 1 d Opht. 1 d Retina RPE Chorioc. Choroid
2.5 0 5 100 J NV 1 h NV 1 h Intact Thinner Open Open
0 15 100 J NV 1 h NV 1 h Intact Thinner Open Open
2.5 1 5 50 J Hypo Light Intact Dense, thin, higher pigm. 50% occluded 50% occluded
1 15 100 J Hypo Light Intact Dense, thin, higher pigm. 50% occluded 50% occluded
2.5 3 5 50 J Hypo Light Light damage Prolif. Presum. occluded Presum. occluded
3 15 100 J Hypo Light Light damage Prolif. Presum. occluded Presum. occluded
2.5 7 5 100 J Hypo Light Relatively intact Prolif. Occluded Occluded
7 15 100 J Hypo Light Relatively intact Prolif. Occluded Occluded
5.0 0 15 50 J NV 1 h NV 1 h Intact Thinner 50% occluded 50% occluded
0 30 100 J NV 1 h NV 1 h Intact Thinner 50% occluded 50% occluded
5.0 7 15 50 J Hypo Light Light damage Less prolif. Occluded Occluded
7 30 100 J Hypo Light Light damage More prolif. Occluded Occluded
Figure 6.
 
Histology 1 hour after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. Displayed is the transition from healthy tissue (left) to irradiated tissue (right). Note the thrombosis of the big choroidal and the small choriocapillary vessels on the right side. The RPE looks regular on the left but thinner on the right side. The photoreceptors look intact. Magnification, ×400.
Figure 6.
 
Histology 1 hour after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. Displayed is the transition from healthy tissue (left) to irradiated tissue (right). Note the thrombosis of the big choroidal and the small choriocapillary vessels on the right side. The RPE looks regular on the left but thinner on the right side. The photoreceptors look intact. Magnification, ×400.
Figure 7.
 
Histology 7 days after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. This section throughout the whole lesion revealed occluded vessels and intact photoreceptors; however, on the RPE level, proliferation was observed (left side). Magnification, ×400.
Figure 7.
 
Histology 7 days after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. This section throughout the whole lesion revealed occluded vessels and intact photoreceptors; however, on the RPE level, proliferation was observed (left side). Magnification, ×400.
Figure 8.
 
The same parameter as in Figure 6 7days after treatment. Note the nodular proliferations of the RPE in localized areas within the treatment zone. Magnification, ×40.
Figure 8.
 
The same parameter as in Figure 6 7days after treatment. Note the nodular proliferations of the RPE in localized areas within the treatment zone. Magnification, ×40.
Figure 9.
 
Higher magnification of the section in Figure 8 7days after treatment. Nodular RPE proliferation and partly multilayered RPE are visible. Magnification, ×400.
Figure 9.
 
Higher magnification of the section in Figure 8 7days after treatment. Nodular RPE proliferation and partly multilayered RPE are visible. Magnification, ×400.
Figure 10.
 
Histology 1 hour after Tookad-PDT using 2.5 mg/kg dye concentration. Irradiation took place with 100 J/cm2 5 minutes after injection. Photoreceptors stayed intact, but the RPE looked a little thinner than usual. Vessels were still open. Magnification, ×250.
Figure 10.
 
Histology 1 hour after Tookad-PDT using 2.5 mg/kg dye concentration. Irradiation took place with 100 J/cm2 5 minutes after injection. Photoreceptors stayed intact, but the RPE looked a little thinner than usual. Vessels were still open. Magnification, ×250.
Figure 11.
 
One day after irradiation with 2.5 mg/kg Tookad 5 minutes after dye injection at 50 J/cm2. Note the intact photoreceptors but the thin condensed and irregular RPE layer. Some of the choriocapillary vessels were occluded but most of the choroidal vessels were still open. Magnification, ×400.
Figure 11.
 
One day after irradiation with 2.5 mg/kg Tookad 5 minutes after dye injection at 50 J/cm2. Note the intact photoreceptors but the thin condensed and irregular RPE layer. Some of the choriocapillary vessels were occluded but most of the choroidal vessels were still open. Magnification, ×400.
Figure 12.
 
Same irradiation pattern as in Figure 11 , but 3 days after treatment. Most of the choroidal and choriocapillary vessels were occluded. Proliferation on the RPE level was noted and also some irregularities in the photoreceptor layer. Magnification, ×400.
Figure 12.
 
Same irradiation pattern as in Figure 11 , but 3 days after treatment. Most of the choroidal and choriocapillary vessels were occluded. Proliferation on the RPE level was noted and also some irregularities in the photoreceptor layer. Magnification, ×400.
Figure 13.
 
Same irradiation pattern as shown in Figure 11 , but 7 days after treatment. Choroidal and choriocapillary vessels were still occluded. Some proliferation on the RPE levels was noted and also slight damage to the photoreceptor layer was seen. Magnification, ×400.
Figure 13.
 
Same irradiation pattern as shown in Figure 11 , but 7 days after treatment. Choroidal and choriocapillary vessels were still occluded. Some proliferation on the RPE levels was noted and also slight damage to the photoreceptor layer was seen. Magnification, ×400.
Figure 14.
 
Summary of all threshold power levels for choroidal vascular occlusion (thresholds for each drug dose [T] at each treatment interval after dye injection).
Figure 14.
 
Summary of all threshold power levels for choroidal vascular occlusion (thresholds for each drug dose [T] at each treatment interval after dye injection).
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Figure 1.
 
Chemical appearance and excitation spectrum of Tookad, which is a water soluble derivative of the natural pigment bacteriochlorophyll. A Palladium atom is incorporated within the central part of the molecule. The peak with the highest absorption coefficient is at 762 nm. There are additional peaks at 530, 385, and 330 nm.
Figure 1.
 
Chemical appearance and excitation spectrum of Tookad, which is a water soluble derivative of the natural pigment bacteriochlorophyll. A Palladium atom is incorporated within the central part of the molecule. The peak with the highest absorption coefficient is at 762 nm. There are additional peaks at 530, 385, and 330 nm.
Figure 2.
 
Irradiation pattern for the application of the 16 PDT lesions per rabbit eye. The X stands for the marker lesions. The first row of PDT lesions was applied 5 minutes after dye injection, the third row at 15 minutes, the second row at 30 minutes, and the fourth row at 60 minutes. Irradiation took place from 100 to 12 J/cm2 initially.
Figure 2.
 
Irradiation pattern for the application of the 16 PDT lesions per rabbit eye. The X stands for the marker lesions. The first row of PDT lesions was applied 5 minutes after dye injection, the third row at 15 minutes, the second row at 30 minutes, and the fourth row at 60 minutes. Irradiation took place from 100 to 12 J/cm2 initially.
Figure 3.
 
(a) Typical irradiation pattern of PDT lesions assigned to be examined histologically (X, marker lesion; A and B, lesions with the same irradiation parameters); (b) Photograph of lesions 1 day after irradiation.
Figure 3.
 
(a) Typical irradiation pattern of PDT lesions assigned to be examined histologically (X, marker lesion; A and B, lesions with the same irradiation parameters); (b) Photograph of lesions 1 day after irradiation.
Figure 4.
 
Early angiography (a), late angiography (b), and fundus photograph (c) 1 hour after irradiation (10 mg/kg Tookad dye injection). The early-phase angiography revealed hypofluorescence, suggesting choroidal vessel occlusion in three lesions (5 minutes: 100 and 50 J/cm2; 15 minutes: 100 J/cm2). Late-phase angiography revealed additional damage by leakage in three other lesions. In the fundus photograph a slight whitening of two lesions is visible. Early angiography (d), late angiography (e), and fundus photograph (f) 1 day after irradiation in the same eye. Early and late angiography phases revealed hypofluorescence suggesting choroidal vessel occlusion in the first three lesions of the first three rows. Additional leakage was found for the 12-J/cm2 lesions in the first three rows and additionally for 100 and 50 J/cm2 in the fourth row. Also notable was that the fast pooling effect in the irradiated area enhanced whitening with all but one parameter (12 J/cm2, 60 minutes after dye injection). Early angiography (g), late angiography (h), and fundus photograph (i) 3 days after irradiation in the same eye. Early-phase angiography revealed hypofluorescence suggesting choroidal vessel occlusion in all but two lesions (25 and 12 J/cm2, 60 minutes after dye injection). Fast pooling was still present, as seen in the late-phase angiography. Fundus photograph shows light lesions, with the especially the first two lesions in the first three rows being very pronounced. Early angiography (j) and fundus photograph (k) 7 days after irradiation in the same eye. The fundus photograph reveals hyperpigmentation in the irradiated areas and fading of the whitening. In some areas, a sort of fibrosis occurred. Because of the hyperpigmentation, a blockade of the fluorescein in the angiography appeared, thus leading to hypofluorescence exactly corresponding with the pigmentation. Early angiography (l) and fundus photograph (m) 14 days after irradiation in the same eye. A fundus photograph revealed enhanced hyperpigmentation over the whole area, with corresponding angiographic hypofluorescence.
Figure 4.
 
Early angiography (a), late angiography (b), and fundus photograph (c) 1 hour after irradiation (10 mg/kg Tookad dye injection). The early-phase angiography revealed hypofluorescence, suggesting choroidal vessel occlusion in three lesions (5 minutes: 100 and 50 J/cm2; 15 minutes: 100 J/cm2). Late-phase angiography revealed additional damage by leakage in three other lesions. In the fundus photograph a slight whitening of two lesions is visible. Early angiography (d), late angiography (e), and fundus photograph (f) 1 day after irradiation in the same eye. Early and late angiography phases revealed hypofluorescence suggesting choroidal vessel occlusion in the first three lesions of the first three rows. Additional leakage was found for the 12-J/cm2 lesions in the first three rows and additionally for 100 and 50 J/cm2 in the fourth row. Also notable was that the fast pooling effect in the irradiated area enhanced whitening with all but one parameter (12 J/cm2, 60 minutes after dye injection). Early angiography (g), late angiography (h), and fundus photograph (i) 3 days after irradiation in the same eye. Early-phase angiography revealed hypofluorescence suggesting choroidal vessel occlusion in all but two lesions (25 and 12 J/cm2, 60 minutes after dye injection). Fast pooling was still present, as seen in the late-phase angiography. Fundus photograph shows light lesions, with the especially the first two lesions in the first three rows being very pronounced. Early angiography (j) and fundus photograph (k) 7 days after irradiation in the same eye. The fundus photograph reveals hyperpigmentation in the irradiated areas and fading of the whitening. In some areas, a sort of fibrosis occurred. Because of the hyperpigmentation, a blockade of the fluorescein in the angiography appeared, thus leading to hypofluorescence exactly corresponding with the pigmentation. Early angiography (l) and fundus photograph (m) 14 days after irradiation in the same eye. A fundus photograph revealed enhanced hyperpigmentation over the whole area, with corresponding angiographic hypofluorescence.
Figure 5.
 
Probability of choroidal vessel occlusion versus the applied radiance exposure for a drug dose of 5 mg/kg. Irradiation took place 15 minutes after injection of the dye. The ED50 threshold for this parameter was 22.8 J/cm2.
Figure 5.
 
Probability of choroidal vessel occlusion versus the applied radiance exposure for a drug dose of 5 mg/kg. Irradiation took place 15 minutes after injection of the dye. The ED50 threshold for this parameter was 22.8 J/cm2.
Figure 6.
 
Histology 1 hour after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. Displayed is the transition from healthy tissue (left) to irradiated tissue (right). Note the thrombosis of the big choroidal and the small choriocapillary vessels on the right side. The RPE looks regular on the left but thinner on the right side. The photoreceptors look intact. Magnification, ×400.
Figure 6.
 
Histology 1 hour after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. Displayed is the transition from healthy tissue (left) to irradiated tissue (right). Note the thrombosis of the big choroidal and the small choriocapillary vessels on the right side. The RPE looks regular on the left but thinner on the right side. The photoreceptors look intact. Magnification, ×400.
Figure 7.
 
Histology 7 days after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. This section throughout the whole lesion revealed occluded vessels and intact photoreceptors; however, on the RPE level, proliferation was observed (left side). Magnification, ×400.
Figure 7.
 
Histology 7 days after irradiation with 5 mg/kg Tookad, 15 minutes after dye injection with 50 J/cm2. This section throughout the whole lesion revealed occluded vessels and intact photoreceptors; however, on the RPE level, proliferation was observed (left side). Magnification, ×400.
Figure 8.
 
The same parameter as in Figure 6 7days after treatment. Note the nodular proliferations of the RPE in localized areas within the treatment zone. Magnification, ×40.
Figure 8.
 
The same parameter as in Figure 6 7days after treatment. Note the nodular proliferations of the RPE in localized areas within the treatment zone. Magnification, ×40.
Figure 9.
 
Higher magnification of the section in Figure 8 7days after treatment. Nodular RPE proliferation and partly multilayered RPE are visible. Magnification, ×400.
Figure 9.
 
Higher magnification of the section in Figure 8 7days after treatment. Nodular RPE proliferation and partly multilayered RPE are visible. Magnification, ×400.
Figure 10.
 
Histology 1 hour after Tookad-PDT using 2.5 mg/kg dye concentration. Irradiation took place with 100 J/cm2 5 minutes after injection. Photoreceptors stayed intact, but the RPE looked a little thinner than usual. Vessels were still open. Magnification, ×250.
Figure 10.
 
Histology 1 hour after Tookad-PDT using 2.5 mg/kg dye concentration. Irradiation took place with 100 J/cm2 5 minutes after injection. Photoreceptors stayed intact, but the RPE looked a little thinner than usual. Vessels were still open. Magnification, ×250.
Figure 11.
 
One day after irradiation with 2.5 mg/kg Tookad 5 minutes after dye injection at 50 J/cm2. Note the intact photoreceptors but the thin condensed and irregular RPE layer. Some of the choriocapillary vessels were occluded but most of the choroidal vessels were still open. Magnification, ×400.
Figure 11.
 
One day after irradiation with 2.5 mg/kg Tookad 5 minutes after dye injection at 50 J/cm2. Note the intact photoreceptors but the thin condensed and irregular RPE layer. Some of the choriocapillary vessels were occluded but most of the choroidal vessels were still open. Magnification, ×400.
Figure 12.
 
Same irradiation pattern as in Figure 11 , but 3 days after treatment. Most of the choroidal and choriocapillary vessels were occluded. Proliferation on the RPE level was noted and also some irregularities in the photoreceptor layer. Magnification, ×400.
Figure 12.
 
Same irradiation pattern as in Figure 11 , but 3 days after treatment. Most of the choroidal and choriocapillary vessels were occluded. Proliferation on the RPE level was noted and also some irregularities in the photoreceptor layer. Magnification, ×400.
Figure 13.
 
Same irradiation pattern as shown in Figure 11 , but 7 days after treatment. Choroidal and choriocapillary vessels were still occluded. Some proliferation on the RPE levels was noted and also slight damage to the photoreceptor layer was seen. Magnification, ×400.
Figure 13.
 
Same irradiation pattern as shown in Figure 11 , but 7 days after treatment. Choroidal and choriocapillary vessels were still occluded. Some proliferation on the RPE levels was noted and also slight damage to the photoreceptor layer was seen. Magnification, ×400.
Figure 14.
 
Summary of all threshold power levels for choroidal vascular occlusion (thresholds for each drug dose [T] at each treatment interval after dye injection).
Figure 14.
 
Summary of all threshold power levels for choroidal vascular occlusion (thresholds for each drug dose [T] at each treatment interval after dye injection).
Table 1.
 
Efficacy of Tookad at Different Time Intervals between Injection and Irradiation in Rabbits That Had Received 5 mg/kg
Table 1.
 
Efficacy of Tookad at Different Time Intervals between Injection and Irradiation in Rabbits That Had Received 5 mg/kg
5 Minutes 15 Minutes 30 Minutes 60 Minutes
100J: 5/5 100% 100J: 5/5 100% 100J: 6/8 75% 100J: 1/5 20%
50J: 8/8 100% 50J: 6/7 86% 50J: 4/10 40% 50J: 0/5 0%
25J: 12/12 100% 25J: 5/10 50% 25J: 2/7 29% 25J: 0/5 0%
12J: 8/11 73% 12J: 2/8 25% 12J: 0/5 0% 12J: 0/5 0%
Table 2.
 
Threshold Power Levels Needed for the Laser Parameters Used to Achieve Angiographically Determined Vessel Occlusion of the Choriocapillaris as Evaluated at Day 3 After Treatment
Table 2.
 
Threshold Power Levels Needed for the Laser Parameters Used to Achieve Angiographically Determined Vessel Occlusion of the Choriocapillaris as Evaluated at Day 3 After Treatment
2.5 mg/kg 5 mg/kg 10 mg/kg
5: 18.8 J/cm2 5: 8.4 J/cm2 5: <8.4 J/cm2
15: 62.0 J/cm2 15: 22.8 J/cm2 15: <8.4 J/cm2
30: >100.0 J/cm2 30: 54.5 J/cm2 30: 11.7 J/cm2
60: — 60: >100.0 J/cm2 60: 54.1 J/cm2
Table 3.
 
Summary of Histologically Examined Laser Lesions Induced by the Different Laser Parameters That Are Capable of Leading to Vessel Occlusion of the Choriocapillaris
Table 3.
 
Summary of Histologically Examined Laser Lesions Induced by the Different Laser Parameters That Are Capable of Leading to Vessel Occlusion of the Choriocapillaris
Drug Dose (mg) Days Post. Treat Parameter Ang. 1 d Opht. 1 d Retina RPE Chorioc. Choroid
2.5 0 5 100 J NV 1 h NV 1 h Intact Thinner Open Open
0 15 100 J NV 1 h NV 1 h Intact Thinner Open Open
2.5 1 5 50 J Hypo Light Intact Dense, thin, higher pigm. 50% occluded 50% occluded
1 15 100 J Hypo Light Intact Dense, thin, higher pigm. 50% occluded 50% occluded
2.5 3 5 50 J Hypo Light Light damage Prolif. Presum. occluded Presum. occluded
3 15 100 J Hypo Light Light damage Prolif. Presum. occluded Presum. occluded
2.5 7 5 100 J Hypo Light Relatively intact Prolif. Occluded Occluded
7 15 100 J Hypo Light Relatively intact Prolif. Occluded Occluded
5.0 0 15 50 J NV 1 h NV 1 h Intact Thinner 50% occluded 50% occluded
0 30 100 J NV 1 h NV 1 h Intact Thinner 50% occluded 50% occluded
5.0 7 15 50 J Hypo Light Light damage Less prolif. Occluded Occluded
7 30 100 J Hypo Light Light damage More prolif. Occluded Occluded
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