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From the Institute of Environmental Engineering, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
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Abstract |
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METHODS. Fertilized chicken eggs were placed under a fluorescence microscope. After intravenous injection of different dyes, time-dependent fluorescence angiography was performed. The effect of PDT parameters was assessed by fluorescence angiography 24 hours after PDT.
RESULTS. Although fluorescence of lipophilic benzoporphyrin derivative monoacid ring A (BPD-MA) remained intravascular during 2 hours, hydrophilic dyes tended to leak through the fenestrated neovascularization. By variation of PDT parameters, vascular damage could be directed toward closure of vessels with a diameter smaller than 10 µm, as measured 24 hours after PDT. High photosensitizer concentrations and high light doses resulted in blood flow stasis within 60 minutes, confirmed by fluorescence angiography.
CONCLUSIONS. Fluorescence angiography and PDT after IV injection into the CAM showed strong similarities to results obtained in clinical tests of PDT in CNV associated with AMD. Thus, this model can provide valuable information about PDT mechanisms and can be used for drug-screening purposes in development of improved sensitizers for the PDT of CNV.
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Introduction |
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Because thermal laser photocoagulation has been shown to cause marked visual loss in patients with subfoveal CNV due to irreversible damage of the retinal pigment epithelium (RPE) and the neural retina,2 3 4 5 6 7 photodynamic therapy (PDT) seems to be an efficient alternative to this treatment modality.
Photodynamic therapy (PDT) of choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD) is based on the interaction of a systemically applied photosensitizing agent with molecular oxygen after irradiation with light of an appropriate wavelength. The initial reaction, leading to reactive singlet oxygen, activates a cascade of chemical and physiological responses, which results in a temporary or permanent occlusion of the irradiated neovascularization.8
Phase I/II and III multicenter trials in Europe and the United States9 10 11 with liposome-encapsulated benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) pioneered this approach. Another second-generation photosensitizer, lutetium-texaphyrin (LU-TEX) underwent a phase I/II clinical study in Lausanne.12 Currently, tin ethyl etiopurpurin (SnET2)13 is under clinical evaluation. Photosensitizers, including phthalocyanines,14 15 16 17 rose Bengal,18 ATX-S10,19 monoaspartylchlorin e6,20 21 and possibly several others are under preclinical evaluation in animal models. However, most of the existing photosensitizers have been primarily optimized for their use in cancer treatment and not for the selective destruction of proliferating neovascularization in CNV associated with AMD. It is therefore a matter of speculation to what extent these drugs are optimal for photodynamic treatment of this disease. To enhance the selective delivery to neovascular tissue through the low-density lipoprotein (LDL) receptor found in abundance on proliferating epithelia, BPD-MA has been premixed with LDLs or encapsulated in liposomes. Such formulations have been tested in multiple animal models, such as CNV in rabbits,22 rabbit pigmented melanoma,23 and CNV in monkeys.24 25 However, although BPD-MA, formulated in liposomes has been shown clinically to be transferred to a significant extent to human LDL, nonselective destruction of normal choroidal capillaries has been observed in clinical PDT.9 10
In view of these drawbacks and the intense interest in finding more powerful treatments for this devastating disease with an incidence of approximately 500,000 new cases annually in Europe and the United States, research in this area will focus on the development of new and more selective photosensitizers for this application. Managing the potentially large number of new photosensitive drugs for the treatment of CNV associated with AMD seems to be difficult, with no efficient in vivo drug-screening procedure for vascular PDT effects.
Because of the complicated mechanisms of vascular photothrombosis, cell culture studies may not respond to mayor questions in this context. Thus, animal models are used14 15 16 17 18 26 27 28 for this purpose. These models, however, are expensive and time consuming and require extensive data acquisition or have no relevance.
Recently, the chick chorioallantoic membrane (CAM) model has been used as an in vivo assay for PDT,29 30 31 32 33 but topical application or intraperitoneal (IP) injection is not relevant to the use of PDT to treat CNV associated with AMD in the clinical setting, in which the photosensitizer is generally administered intravenously (IV). The CAM has received considerable attention in angiogenic research. It is the major respiratory organ of the chick embryo. The well-vascularized surface is formed by the fusion of two mesodermal layers and has several advantages as a model for PDT. The CAM assay is easily accessible, inexpensive, and easy to handle. Because of the transparency of its superficial layers, nearly any wavelength in the visible part of the electromagnetic spectrum can be used for fluorescence analysis; visualization, and documentation are facilitated. When normally developing embryos are used, it is not clear whether the CAM model can distinguish between proliferating and nonproliferating blood vessels. However, it has been shown that modulation of the angiogenic activity by exposure to basic fibroblast growth factors can add these aspects to present approaches.34
We present a simple method to assess the relative efficacy of new photosensitive drugs in a vascular system. One goal of the present work was to demonstrate the feasibility of using the CAM model for PDT after intravenous drug delivery in the blood circulation of the embryo and to compare the findings with clinical observations. Furthermore, it was shown that the use of a second injection of a fluorescent dye after PDT is useful in assessing vascular occlusion. This double-injection technique has been shown to be no more complicated than other procedures, such as intraperitoneal administration, but it is rather close to the clinical situation, consisting of PDT and fluorescence angiography.
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Methods |
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CAM Preparation
Fertilized chicken eggs were disinfected, numbered, and
transferred into a static incubator, set at 37°C and 60% humidity.
On embryo development day (EDD) 3 a hole approximately 3 mm in
diameter was drilled into the eggshell and covered with cling film. The
egg was then returned to the incubator until use.
Fluorescence Pharmacokinetics
Low-light fluorescence imaging was performed with an air-cooled,
slow-scan 16 bit CCD camera (EEV P86231; Wright, Endfield, UK) fitted
to a fluorescence microscope (BH2-RFC; Olympus, Tokyo, Japan) equipped
with an objective (S.Plan Apo; Olympus; 4x/0.16; working distance, 9.8
mm). Fluorescence excitation, controlled by an excitation shutter
(Uniblitz Driver D122; Vincent, Rochester, NY), was provided by a 100-W
mercury arc lamp filtered by an interference filter (fluorescein:
ex = 420–490 nm; BPD-MA and R101:
ex = 390–440 nm). Exposure times were
controlled by an electronic shutter and set to 1 second. Before
excitation, light passed through a pinhole of variable diameter defined
the total surface of irradiation. A microscope excitation cube
(Olympus), consisting of a dichroic mirror (DM), a barrier filter (BA;
fluorescein: Olympus cube B = DM515 + O515; BPD-MA and R101:
Olympus cube BV = DM455 + Y475; mTHPC: cube Olympus V = DM455
+ Y475) and a long-pass filter (RG 610; Schott, Mainz, Germany), was
used to suppress excitation light and tissue autofluorescence in the
detection pathway. Digital imaging, data display, and storage were
performed with an IBM-compatible PC connected to the CCD camera.
On EDD 12–13 the hole in the eggshell was extended to a diameter of
approximately 2 to 3 cm. The egg was then placed under the objective of
the microscope using a Büchner funnel rubber dam. Before
dye injection, CAM autofluorescence was recorded using a band-pass
filter ( = 540–580 nm) placed in the emission pathway instead
of a long-pass filter. With the embryos directly under the microscope,
100 µl of the corresponding dye (1 mg/ml) was injected into one of
the principal blood vessels of the CAM (diameter 
300–500 µm), by
using a 33-gauge needle (Hamilton, Reno, NV).
Fluorescence imaging was performed during an 800-second period at
regular time intervals. The time-dependent evolution of the
fluorescence intensity inside the blood vessels
(Iin) in relation to the surrounding
tissue (Iout) was expressed by means
of normalized photographic contrast
(Cphot)
 | (1) |
PDT and Damage Assessment
At stage EDD12–13 the egg was placed under the microscope as
described above. Before injection, a 5-mm sterile Teflon ring was
placed onto the CAM surface at a distance of approximately 10 mm from
the injection site, to demarcate the irradiation site, which contained
blood vessels with a diameter of 5 to 150 µm.
The embryos were injected with 100 µl of freshly prepared BPD-MA solution, containing 0.2, 0.04, and 0.02 mg/ml, respectively. Directly after infusion, a fluorescence image (BPD-MA fluorescence angiogram [BPD-FA]) was obtained as described earlier. The size of the irradiation area was further reduced to a diameter of 1.4 mm, to allow direct comparison between irradiated and nonirradiated blood vessels after PDT. After 60 seconds, the reduced area was irradiated with 7.5, 15, 25, 40, and 50 J/cm2 at 440 nm, a wavelength strongly absorbed by BPD-MA. During irradiation, a second BPD-FA was obtained. Light doses were measured with a calibrated photometer (IL1700 Radiometer; International Light, Newburyport, MA).
To test the long-term stability of liposomal BPD-MA solutions with respect to their PDT efficacy, eggs were injected with 100-µl liposomal solutions containing 0.02 mg/ml BPD-MA, which were reconstituted 2 weeks before use and stored at 4°C. These solutions were shaken for at least 1 minute before administration, and eggs were treated as described.
In a third series, the time interval between injection and irradiation was varied, using a light dose of 25 J/cm2. The corresponding drug light intervals were 1, 3, 6, and 12 minutes. After irradiation, the eggs were covered with blackened Parafilm (Aldrich, Buchs, Switzerland) and returned to the incubator overnight.
Damage assessment was performed by a second injection of R101 24 hours after PDT. For this purpose, the eggs were put in exactly the same position and orientation as during PDT. Subsequently, fluorescence imaging, consisting of autofluorescence and R101-fluorescence angiography (R101-FA), was performed as described earlier. Using this second injection the perfusion of blood vessels was easily documented, because nonperfused vessels showed no intravascular fluorescence. Furthermore, a direct comparison with fluorescence images, taken during PDT, gave quantitative information on the reduction of irradiated blood vessels.
The extent of damage was classified using an arbitrary damage scale between 1 and 5 (see Table 1 ) on the basis of blood vessel destruction or size reduction in comparison with their original size (see Fig. 1 ). Ten eggs were used for each data point.
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Results |
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Figure 2 shows a representative sequence of fluorescence angiograms obtained after injection of R101 into one of the principal chorioallantoic vessels. After infusion of the dye, the chromophore was distributed throughout the entire blood vessel system. Depending on which part of the blood circulating system, arteriolar or venular, received the dye application, some blood vessels and capillaries remained poorly fluorescent for at least 5 seconds. However, after a short dilution period of approximately 10 seconds, the dye appeared homogeneously distributed in the blood vessels of the blood-circulating system of the embryo. Because of leakage of the dye, the background fluorescence increased, and the photographic contrast between blood vessels and the surrounding tissue diminished. At 360 seconds this contrast vanished with R101. After this turnover point, the blood vessels appeared darkened against the highly fluorescent background. This is mainly due to the higher fluorescence yield when xanthene dyes are outside the blood circulation35 and to a lesser extent to reabsorption of the fluorescence by the blood vessels and circulating blood.
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PDT of CAM Blood Vessels
Injection into the vascular system of the embryo has been
found to be highly reproducible according to the pharmacokinetic
studies. After a short dilution period, the BPD-MA fluorescence was
homogeneously distributed, and therefore it can be estimated that
a reproducible PDT effect can be expected as early as 60 seconds after
administration. Furthermore, due to its long retention time in the
embryo’s blood circulation, BPD-MA can be used to induce vascular
damage over an experimentally acceptable time span. To evaluate our
model system as a rapid screening procedure for new
photosensitizers for PDT of CNV associated with AMD, BPD-MA was
chosen as a first test substance, because it allows direct comparison
with clinical results.
In a series of 280 eggs, 10 were found dead 24 hours after reincubation. In Figure 4 a typical sequence, consisting of autofluorescence (Fig. 4A) , BPD-FA (Fig. 4B) , PDT (Fig. 4C) , and R101-FA 24 hours after PDT (Fig. 4D) is shown. Irradiation with 25 J/cm2 at 440 nm and a photosensitizer dose of 4 µg/embryo resulted in total vasoconstriction (damage scale 5) in the illuminated area, as indicated by R101-FA. Under all conditions used, structure as well as the diameter of nonirradiated blood vessels remained unchanged. The sequence, shown in Figure 4 , demonstrates the advantage of our double-injection method. Comparison of nonirradiated with irradiated areas served as a blind probe in the same egg. Furthermore, a determination of the fate of different blood vessels after irradiation is possible, because both fluorescence images are taken using the same illumination conditions.
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Administration of the same dose using liposomal BPD-MA, which was reconstituted 2 weeks before use, did not show any damage to the vascular bed, although fluorescence intensities were similar after infusion (see Fig. 6 2 * µg/embryo).
Because BPD-MA fluorescence intensity diminishes as a function of time after injection, it can be supposed that a variation of the time between infusion and irradiation may change the therapeutic outcome of the treatment. However, with the use of a treatment regimen that was found to be slightly to high (4 µg/embryo, 25 J/cm2), no significant difference was observed between the vascular occlusion efficacy of treatments performed 1 minute or 12 minutes after injection of BPD-MA (see Fig. 7 ).
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Discussion |
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Cell culture experiments are not optimal for obtaining information on the vascular damage efficacy of photodynamic agents.32 Thus, new methods should be developed to provide an effective, low-cost drug-screening system for promising photosensitizers. The CAM assay36 has often been used to study vascular and angiogenic effects in vivo. Although this model has already been used for PDT purposes,29 30 31 32 33 most approaches were not specifically adapted for AMD-PDT. Topical application and IV injection represent significantly different uptake mechanisms (i.e., the usual simple deposition of the photodynamic agent on the CAM surface is quite different from the clinical situation29 31 32 in which intravenous injections are used). The same applies to IP administration.29
The present study shows a modification of the CAM model to make it serve as a low-cost, simple, informative, and appropriate assay for the prediction of photosensitizer characteristics related to PDT-induced vascular damage encountered in clinical tests. To evaluate the modified CAM procedure for the assessment of new photoactive drugs in AMD-PDT, it is essential to draw a comparison to clinical aspects. For this purpose, among other observations, two important properties in PDT for CNV in the human eye are suitable. First, the leakage out of the CNV of the dye after intravenous injection, and second the response to PDT in closing the CNV.
The time dependence of the fluorescence of the sensitizer inside and outside the blood vessels is of importance for two principle reasons. First, fast leakage of small, water-soluble dyes, like fluorescein, through fenestrated neovessels is frequently used for the diagnosis of CNV in the human eye.9 10 Note that this technique has been used to quantitatively monitor the effect of PDT treatments of this disease.37 Second, a photodynamic agent should not leak out of these fenestrated neovascular structures and choriocapillaries too quickly, to prevent photodynamic action on neighboring structures such as the RPE or the photoreceptors.19
The results from the fluorescence pharmacokinetics measurements in our modified CAM model permit observation of the relations between the leakage of neovessels observed in the wet form of AMD, in which leaking fluorescein demarcates CNV in the early phase of the fluorescence angiographies.
Husain et al.24 have studied the leakage of BPD-MA in experimental neovascularization induced in the iris of cynomolgus monkeys. They found only minimal leakage but a significant decrease of vascular BPD-MA fluorescence intensity within 10 minutes. Although most of the BPD-MA was associated with blood lipoproteins among which are LDLs, they found no differences between the fluorescence pharmacokinetics of neovessels and that of normal vascularization. This is in agreement with our observations that the fluorescence time profiles are similar for blood vessels of different diameter.
One critical point in the search of a valid new drug-screening
procedure for AMD-PDT is to establish an at least semiquantitative
relation with clinical results. Thus, in our experimental setup we
deliberately chose conditions that were close to those used in the
clinic for drug administration and damage assessment. To evaluate
vascular damage in our model, we injected BPD-MA, for which
considerable clinical information is available. Using this
photosensitizer, a drug dose of 6 mg/m2
corresponding to approximately 0.17 mg/kg for an average adult, is
injected. Light doses of 50 J/cm2 were used,
because higher light doses clinically have closed retinal vessels and
have impaired the patient’s visual acuity.9
Supposing a
typical diameter of approximately 30 µm of vessels occurring in
CNV,38
a successful AMD PDT with BPD-MA would be ranked 3
on our arbitrary damage scale. In our model this value was achieved
using a photosensitizer dose of 4 µg/embryo and a light dose of 15
J/cm2 or a photosensitizer dose of 2 µg/embryo
and a light dose of 40 J/cm2 (Fig. 6)
. Based on
the mean weight estimates for embryos39
on EDD13 (13 g)
this corresponds to photosensitizer doses of 0.3 and 0.15 mg/kg,
respectively. In clinical practice, however, irradiation is performed
at 690 nm, in which absorption of BPD-MA is approximately 2.5 times
smaller than at 440 nm. This should be taken into account in subsequent
animal tests in which other irradiation wavelength may be more
favorable. In contrast, in the CAM, inner filters, such as melanin in
the human RPE, that prevent the light in the blue spectral region from
reaching the deeper choroidal vasculature are absent. Thus, it is
possible to work with excitation light of approximately 440 nm, which
enables use of standard epifluorescence microscopes, providing fluence
rates in these wavelength ranges similar to those used in clinics. In
biologic systems the internal conversion from the S2 to the S1 state is
rapid, compared with processes such as fluorescence or intersystem
crossing. Thus, using 440 nm instead of 690 nm increases the total
number of excited photosensitizer molecules but does not influence the
fluorescence or singlet oxygen quantum yields.
To the best of our knowledge there are at present no data regarding the stability of liposomal formulated BPD-MA. For this purpose we included a series of embryos, injected with previously prepared BPD-MA solutions in our study protocol. Although fluorescence intensities as well as the fluorescence distribution after IV injection under these conditions were found to be similar to those of freshly prepared BPD-MA solution, we observed significantly reduced phototoxicity. Whether this was due to the instability of the liposomes containing egg phosphatidylglycerol and phosphatidylcholine40 41 42 and thus reduced binding to endothelial structures43 or to the degradation of the photosensitizer itself requires additional investigations.
To assess reproducibility of our model, we used an extraordinary high number of eggs per data point. However, because of the small variations observed under our experimental conditions, it is probable that the total number of eggs can be significantly reduced for a preliminary evaluation of the efficacy of photosensitizers.
The presented bioassay provides useful information for assessing vascular damage after photodynamic treatment. Because of the architecture of the CAM, it is not clear to what extent the present model addresses the selectivity of new photosensitizers, because established vessels such as those in human CNV are absent. As mentioned earlier, however, the CAM model may easily be extended by the gel–nylon mesh technique44 or the gelatin sponge assay,34 which profits from newly formed blood vessels perpendicularly grown to the plane of the CAM into the graft, which does not contain preexisting vessels.
Unfortunately, other structures of interest in the treatment of CNV associated with AMD such as the RPE, Bruch’s membrane, or the neural retina are not present in our model. Again, the model is not intended to completely replace animal models for in vivo testing of photosensitizer selectivity, but it will help to minimize the number of time-consuming and expensive animal tests.
Finally, the present use of the CAM model offers the advantage of easy damage assessment performed by R101-FA 24 hours after PDT. This supplementary injection of a fluorescing dye enables observation of the blood flow through irradiated vessels33 and the collection of quantitative information on the real-time status of the blood circulation within the irradiated area, compared with nonirradiated zones. The recording of digital fluorescence image before, during, and 24 hours after irradiation allows at least semiquantitative evaluation of vascular PDT effects using standard imaging processing software. Even, a follow-up longer than 1 day (up to 2–3 days) can be performed, when necessary. Because both irradiated and nonirradiated areas are available in one fluorescence image, numerical evaluation can provide information on PDT effects of the proliferation pattern of the capillary network, blood vessel development, and other factors related to angiogenic processes. Furthermore, a direct, quantitative assessment of photothrombic effect due to PDT is given by numerical comparison BPD-FA images during PDT.
In conclusion, we have demonstrated that this new technique, consisting of IV injection and subsequent fluorescence angiography, transforms the CAM assay into a model that is appropriate for rapid, inexpensive drug screening of photosensitizers for vascular effects. It has been shown to correlate well with clinical observations and will be a major aid in the selection of promising new for PDT of CNV associated with AMD. Furthermore, it is sufficiently close to clinical reality that it appears to be able to predict treatment regimens, which will considerably reduce the number of animals necessary for clinical testing during the late stages of drug development.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 9, 2000; revised August 22, 2000; accepted September 15, 2000.
Commercial relationships policy: N.
Corresponding author: Norbert Lange, Swiss Federal Institute of Technology, Institute of Environmental Engineering, DGR-LPAS, CH-1015 Lausanne, Switzerland. norbert.lange{at}epfl.ch
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