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/cm
2; 50 J/cm
2), 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 10
5);
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.
Efficacy of Tookad Regarding Different Time Intervals between Injection and Irradiation
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/cm
2 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/cm
2 and relatively light doses of 5 to 20 J/cm
2. 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/cm
2 at an irradiance of 600 mW/cm
2(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/cm
2 in pigmented rabbits and above 0.88 J/cm
2 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 10
5. 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.
Supported by Negma-Lerads, Magny-Les-Hameaux, France.
Submitted for publication May 15, 2006; revised June 27 and July 17, 2006; accepted September 11, 2006.
Disclosure:
C. Framme, Negma-Lerads (F);
H.G. Sachs, Negma-Lerads (F);
B. Flucke, Negma-Lerads (F);
D. Theisen-Kunde, Negma-Lerads (F);
R. Birngruber, Negma-Lerads (F)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Carsten Framme, University Eye Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany;
[email protected].
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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