 |
|
Selective Photodynamic Therapy by Targeted Verteporfin Delivery to Experimental Choroidal Neovascularization Mediated by a Homing Peptide to Vascular Endothelial Growth Factor Receptor-2
Reem Z. Renno, MD;
Yoshiko Terada, MD;
Makhluf J. Haddadin, PhD;
Norman A. Michaud, MS;
Evangelos S. Gragoudas, MD;
Joan W. Miller, MD
Arch Ophthalmol. 2004;122:1002-1011.
ABSTRACT
| |
Objective To evaluate the feasibility, efficacy, and selectivity of photodynamic therapy (PDT) using targeted delivery of verteporfin to choroidal neovascularization (CNV) in the rat laser-injury model of CNV.
Methods We performed PDT in rat eyes on experimental CNV and normal retina and choroid using verteporfin conjugates. A targeted verteporfin conjugate was made by conjugating verteporfin (after isolation from its liposomal formulation) to a modified polyvinyl alcohol (PVA) polymer (verteporfin-PVA) followed by linkage to the peptide ATWLPPR known to bind the receptor for vascular endothelial growth factor, VEGFR2. The verteporfin-PVA conjugate served as a control. We performed fluorescent fundus angiography to determine the optimal timing of light application for PDT using the conjugates. Closure of CNV was assessed angiographically and graded in a masked standardized fashion. We used standardized histological grading to compare the effects on normal retina and choroid.
Results The verteporfin-PVA conjugation ratio was on average 28:1. The conjugate retained typical emission/excitation spectra and photosensitizing activity and was as efficient as an equivalent amount of verteporfin. Peak intensity of targeted verteporfin in CNV was detected angiographically at 1 hour after intravenous injection. Photodynamic therapy using targeted verteporfin (3 or 4.5 mg/m2) with light application 1 hour after drug injection showed angiographic closure of all treated CNV (17/17) 1 day after treatment. Photodynamic therapy using verteporfin-PVA at the same drug dose achieved closure in 18 of 20 CNV. Histological examination after PDT of normal retina and choroid using targeted verteporfin and irradiation at 1 hour showed minimal effect on retinal pigment epithelium and no injury to photoreceptors, whereas PDT using verteporfin-PVA resulted in retinal pigment epithelium necrosis and mild damage to photoreceptors.
Conclusions Verteporfin bound to the targeting peptide, ATWLPPR, retained its spectral and photosensitizing properties. Angiography demonstrated localization of the targeted verteporfin 1 hour after injection. Photodynamic therapy using targeted verteporfin and the control conjugate were more effective in causing CNV closure than standard liposomal verteporfin. The targeted verteporfin resulted in more selective treatment than the control conjugate or standard verteporfin. These results suggest that targeted PDT strategies based on selective expression of receptors on CNV vasculature may improve current therapy.
Clinical Relevance Targeted PDT for CNV is feasible and may offer a qualitative improvement in current treatments for patients with age-related macular degeneration. This study provides the basis for further preclinical studies of targeted PDT strategies and subsequent clinical trials.
INTRODUCTION
Photodynamic therapy (PDT) using verteporfin as a photosensitizer has been demonstrated in large clinical trials to be an effective new treatment for subfoveal choroidal neovascularization (CNV) secondary to age-related macular degeneration and other causes.1-4 The preferential occlusion of CNV after PDT as currently practiced is based on the differences in biodistribution of the photosensitizer between CNV and retinal vessels at the time that light is applied. Preclinical work on PDT with verteporfin (QLT PhotoTherapeutics Inc, Vancouver, British Columbia) has shown treatment-related damage to the surrounding retina, choroid, and retinal pigment epithelium (RPE).5-7 This collateral damage is cumulative with repeated PDT.7-8 Modifications to PDT, including combination with antiangiogenic therapy9-10 or with targeted photosensitizer, may improve selectivity and vision outcomes.
Homing peptides are an emerging class of pharmaceuticals that exploits differences between cell types by binding specific cell membrane receptors.11-13 The peptide targeting of photosensitizers might enable specific and enhanced retention of photosensitizer to CNV and allow more selective PDT with minimal adverse effects.
Vascular endothelial growth factor (VEGF) expression and binding of VEGF to its kinase domain receptor (KDR/FLK1 or VEGFR-2) is an important mediator of angiogenesis, including retinal and choroidal neovascularization.14-15 Inhibition of VEGF and VEGFR-2 prevents retinal and choroidal neovascularization.16-18 The presence of KDR or VEGFR-2 has been demonstrated in normal vessels but shows increased expression in endothelial cells of neovascular tissue and is thus a potential candidate for peptide-mediated targeting of CNV.19-20 The peptide ATWLPPR is reported to specifically bind VEGFR-2 and completely inhibit binding of native VEGF, thereby preventing VEGF-induced angiogenesis in vivo.21
We propose to use ATWLPPR as a homing peptide bound to verteporfin to target verteporfin to CNV by binding to VEGFR-2 on CNV. Because VEGFR-2 is overexpressed on neovascular endothelium, normal vessels should be relatively spared and retinal cells should be unaffected after PDT using verteporfin targeted to VEGFR-2. Experiments were designed to evaluate the efficacy and selectivity of PDT with VEGFR2-targeted verteporfin in the rat laser-injury model of CNV.
METHODS
ISOLATION OF FREE VERTEPORFIN FROM ITS LIPOSOMAL FORMULATION
We recovered verteporfin at a concentration of 2 mg/mL in liposomal formulation from material prepared for clinical treatments; leftovers were refrigerated and processed within 2 weeks to ensure activity.22 Liposomal verteporfin was acidified using a 6M hydrochloric acid solution, and separation of organic (verteporfin) and aqueous (liposome) layers was achieved using dichloromethane (CH2Cl2). After concentrating the solution by means of evaporation, verteporfin was further purified by means of gravity chromatography on silica gel using an eluting solvent consisting of a 3:1 ratio of CH2Cl2 to methanol. The verteporfin solution was evaporated to dryness and redissolved in dimethyl sulfoxide.
SYNTHESIS OF VEGFR-2–TARGETED VERTEPORFIN
Previous studies linking photosensitizers to antibodies suggested that derivatives of polyvinyl alcohol (PVA; molecular weight, 10 000-11 000 Da; Sigma-Aldrich Corp; St Louis, Mo) provide suitable carriers for photosensitizers without jeopardizing the biological activity of the photosensitizer.23 The procedure for loading verteporfin on PVA has been described elsewhere.23-24 Briefly, PVA was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate (Sigma-Aldrich Corp) and 1,6-hexanedimanine (Sigma-Aldrich Corp) to produce side chains containing terminal-free amino groups. Conjugation of modified PVA with verteporfin was affected by reacting it with a 25-fold molar excess of verteporfin in the presence of carbodiimide as coupling agent in dimethyl sulfoxide. Carrier conjugates were analyzed by means of high-performance liquid chromatography using a column with ultrasphere consisting of 5-µm optical density, 250 x 4.6 mm; a solvent system consisting of solution A (500 mL each of 1% [NH4]2SO4 and CH3CN and 50 mL of CH3COOH) and solution B (500 mL of 1% [NH4]2 SO4 and C4H8O and 50 mL of CH3COOH 50mL); a flow rate of 1.7 mL/min; and a gradient of solutions A-B of 60%:40% for 5 minutes, then a starting gradient flow from 40% solution B to 70% solution B in 20 minutes and staying at 70% solution B for 5 minutes before returning to 40% solution B. Verteporfin-PVA eluted at 8 to 10 minutes, whereas unconjugated verteporfin eluted at 18 to 19 minutes. The molecular weight of verteporfin-PVA was determined by mass spectrometry and found to be approximately 28 kDa (verteporfin-PVA ratio, approximately 28:1).
Before binding to the homing peptide, thiol groups were introduced to verteporfin-PVA using 3-mercaptopropionic acid (Sigma-Aldrich Corp) in acetate buffer (pH, 5.5). Coupling of verteporfin-PVA to the targeting peptide (ATWLPPR; molecular weight, 840 Da synthesized to our specifications by Anaspec, San Diego, Calif) was performed using sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester as a heterobifunctional cross-linking reagent in carbonate buffer (pH, 8.5). Products were separated by means of high-performance liquid chromatography with the solvent system of solution A, 100% water, and 0.1% trifluoroacetic acid and solution B, 100% acetonitrile and 0.1% trifluoroacetic acid (gradient starting at solutions A-B, 80%:20% and going to 80% solution B in 45 minutes). The molecular weight of VEGFR-2–targeted verteporfin was determined by mass spectrometry and found to be approximately 30 kDa. For the remainder of this report, the photosensitizer dose will be expressed in verteporfin-equivalents (in milligrams divided by the square of the body surface in square meters) as determined by spectrofluorometry using a verteporfin calibration curve. Briefly, a calibration curve correlating concentration vs spectral emission of liposomal verteporfin was constructed. Spectral emission of a sample of VEGFR-2–targeted verteporfin was determined and used to extrapolate from the calibration curve its verteporfin content.
All intermediates (free verteporfin, verteporfin-PVA, and VEGFR-2–targeted verteporfin) were found to have the same excitation and emission spectra as the liposomal verteporfin formulation as determined by spectrofluorometry and to preserve an equivalent in vitro photosensitizing activity (tested in human umbilical vein endothelial cells) as determined by the tetrazolium salt MMT assay.25
EXPERIMENTAL CNV MODEL
The rat laser-injury model of CNV was modified from earlier reports and used in our laboratory for PDT.26-28 Adult male pigmented rats (Brown-Norway; Charles River Laboratories, Wilmington, Mass) were used in the study, and all procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Research and the guidelines of the Massachusetts Eye and Ear Infirmary (Boston) Animal Care Committee. The rats were anesthetized for all procedures with an intramuscular injection of 0.2 mL of a 50:50 mixture of ketamine hydrochloride (20 mg/mL) and xylazine hydrochloride (100 mg/mL) (both from Phoenix Pharmaceutical Inc, St Joseph, Mo). For killing, a mixture consisting of pentobarbital sodium, 390 mg/mL; propylene glycol; ethanol; and water (Fatal Plus, 650 mg/kg; Vortech Pharmaceuticals, Dearborn, Mich) was given intraperitoneally.
The pupils were dilated with 5% phenylephrine hydrochloride and 0.8% tropicamide, and 4 to 6 photocoagulation lesions using a Coherent 920 argon dye laser (100-µm spot size; 0.1-second duration; 630 nm and 120-160 mW) (Coherent Medical Laser, Palo Alto, Calif) were delivered between the retinal vessels in a peripapillary distribution in each fundus using a slitlamp delivery system and a cover glass as a contact lens. Production of a bubble at the time of laser confirmed the rupture of the Bruch membrane. The presence of CNV was confirmed by fluorescein angiography using a TRC-50VT camera (Topcon, Paramus, NJ) with images captured on IMAGEnet for Windows system (Topcon) after an injection of 1 mL of 1% fluorescein sodium (Alcon, Fort Worth, Tex). A choroidal neovascular membrane was defined as present if early hyperfluorescence with late leakage was present at the site of the inducing laser injury as previously described.28
PHOTOSENSITIZERS
Verteporfin conjugates were tested in eyes with experimental CNV and in normal eyes. For targeted PDT, verteporfin-PVA was bound to the peptide ATWLPPR. The untargeted photosensitizer conjugate, verteporfin-PVA, was chosen as a control because both molecules have comparable molecular weights (28 and 30 kDa, respectively). The results with the targeted and control conjugates were compared with previous results in the rat model with PDT using verteporfin (718 Da) in liposomal formulation, which is the therapy currently in clinical use.28
VEGFR-2–TARGETED VERTEPORFIN AND VERTEPORFIN-PVA ANGIOGRAPHY
Angiographies with VEGFR-2–targeted verteporfin and verteporfin-PVA were performed using a standard fundus camera but with verteporfin-specific filters, with the excitation spectral band centered at 580 nm and fluorescence detection at 695 nm. Maximal gain settings were required. Photosensitizer doses were given via tail-vein injection, and angiography was performed at a drug dose of 12 mg/m2. Conversion to body surface area (in square meters) from weight (in kilograms) was made using a nomogram developed by Gilpin.29 Relative fluorescence intensities were determined by visual analysis of the angiograms.
PDT IN A RAT CNV MODEL
Photodynamic therapy was performed on experimental CNV and areas of normal choroid and retina. Laser light of 689 nm was administered using a diode laser (Coherent Medical Laser, Palo Alto, Calif) delivered via a slitlamp adapter (Laserlink; Coherent Medical Laser). Laser power at the focal plane was measured with a power meter (Coherent Fieldmaster; Coherent, Auburn, Calif). The laser spot size was set at 750 µm and was confirmed using a micrometer, and the irradiance used was 600 mW/cm2, which was delivered for 17, 42, or 83 seconds to achieve total energy doses of 10, 25, or 50 J/cm2, respectively. Activating light fluences were based on previous dosimetry established in the rat model of CNV for verteporfin PDT.28
FIRST OUTCOME MEASURE: CNV CLOSURE
Fluorescein angiograms were performed at 24 hours after treatment. Closure of CNV was defined by absence of leakage from CNV compared with the baseline angiogram as previously described. All angiograms were graded in masked fashion (as to dose and photosensitizer) by 2 experienced graders (E.S.G. and J.W.M.).
HISTOLOGICAL EVALUATION
Eyes were enucleated and the lens and anterior segment were removed. The remaining eyecups were placed in a fixative containing 2.5% glutaraldehyde and 2% formaldehyde in 0.1M cacodylate buffer (pH, 7.4) at 4°C overnight. Tissue samples were then postfixed in 2% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in epoxy resin. For light microscopy, 1-µm sections were stained with 1% toluidine blue in 1% borate buffer and examined with a Zeiss photomicroscope (Axiophot, Oberkochen, Germany). For electron microscopy, sections were stained with a saturated aqueous uranyl acetate solution and Sato lead stain. Sections were viewed with a transmission electron microscope (Philips CM 10; Royal Philips Electronics, Eindhoven, the Netherlands).
SECOND OUTCOME MEASURE: EFFECT ON NORMAL CHOROID AND RETINA
Grading of sections was performed in a masked fashion (as to dose and photosensitizer) by an experienced grader (N.A.M.) using the following histological grading scheme for PDT effects on normal choroid and retina modified from Kramer et al6: grade 1 indicates damage in the RPE and photoreceptors, with occasional pyknosis in the outer nuclear layer (ONL), with or without choriocapillaris damage; grade 2, choriocapillaris closure, RPE and photoreceptor damage, and 10% to 20% pyknosis in the ONL; grade 3, grade 2 with less than 50% pyknosis in the ONL; grade 4, grade 3 with greater than 50% pyknosis in the ONL; and grade 5, grade 4 with damage to large choroidal vessels or retinal vessels or inner retinal layers.
RESULTS
TEMPOROSPATIAL LOCALIZATION OF TARGETED VERTEPORFIN
To test the temporospatial localization of targeted and control verteporfin conjugates in the retinal and choroidal circulations and CNV, angiography was performed using targeted verteporfin and verteporfin-PVA at a dose of 12 mg/m2. At the earliest time points captured (5-10 seconds) after drug injection, fluorescence of targeted and control verteporfin conjugates was already noted in the choroidal and retinal circulation. Fluorescence intensity peaked in the retinal circulation at approximately 30 to 60 seconds and then cleared from retinal circulation (20-21 minutes), followed by the choroidal circulation (35-40 minutes). The drug was retained within the CNV with peak intensity of fluorescence seen at 1 hour (Figure 1). Verteporfin-PVA exhibited the same temporospatial localization. In contrast, liposomal verteporfin in CNV peaked at 15 to 20 minutes.28
|
|
|
|
Figure 1. Targeted verteporfin angiography in choroidal neovascularization (CNV). A, A typical fluorescein angiogram performed 2 to 3 weeks after the laser induction of the experimental choroidal neovascular membranes and before photodynamic therapy shows early hyperfluorescence. B, Another view shows late leakage corresponding to CNV. C, Angiography of the same eye using targeted verteporfin (12 mg/m2) shows peak intensity in the retinal vasculature at approximately 30 seconds after injection. D, Peak intensity of targeted verteporfin in CNV at 1 hour after injection (arrowheads).
|
|
|
EFFICACY OF PDT WITH TARGETED VERTEPORFIN
Since angiography demonstrated accumulation of targeted verteporfin and verteporfin-PVA in CNV at 1 hour after drug administration, this was the time selected for light application for PDT. Photodynamic therapy was performed using targeted verteporfin or verteporfin-PVA on 27 or 32 areas of CNV, respectively. Angiographic closure of CNV was assessed at 24 hours after PDT and was defined as an absence of leakage from the CNV compared with the pretreatment fluorescein angiogram. The angiograms were graded with masking as to light, drug dose, and photosensitizer used. Twenty-four hours after PDT with verteporfin-PVA, occluded CNV typically showed a circle of hypofluorescence in the early frames corresponding to the treatment spot, with late leakage from injured RPE and choriocapillaris usually originating at the rim of the treatment spot (Figure 2). Open CNV showed early hyperfluorescence in the area of CNV and late leakage. The same pattern was observed with PDT with verteporfin or with targeted verteporfin irradiated at 15 to 20 minutes (optimal timing for verteporfin but suboptimal timing of treatment for targeted verteporfin). In contrast, CNV treated with PDT with targeted verteporfin and activating light applied at 1 hour showed no early hypofluorescence and only rarely any late hyperfluorescence or leakage. In addition, occluded CNV did not show any hyperfluorescence or late leakage in the area of CNV, consistent with occlusion of CNV (Figure 3).
|
|
|
|
Figure 2. Photodynamic therapy (PDT) for choroidal neovascularization (CNV) using verteporfin conjugated to a modified polyvinyl alcohol polymer (PVA). A, Early-phase pretreatment fluorescein angiogram shows hyperfluorescence in areas of CNV. B, Another view shows increasing hyperfluorescence and leakage of CNV. C, Early phase of fluorescein angiogram 24 hours after PDT using verteporfin-PVA with 4.5 mg/m2 and laser fluences of 10 (arrowheads), 25 (arrows), or 50 (asterisk) J/cm2 shows hypofluorescence corresponding to the treatment spots. D, Late-phase fluorescein angiogram shows hyperfluorescence (asterisk) and leakage that originated at the rim of the treatment spots (arrowheads and arrows).
|
|
|
|
|
|
|
Figure 3. Photodynamic therapy (PDT) for choroidal neovascularization (CNV) using targeted verteporfin. A, Early-phase pretreatment fluorescein angiogram shows hyperfluorescence in areas of CNV. B, Another view shows increasing hyperfluorescence and leakage from CNV. C, Early-phase fluorescein angiogram 24 hours after PDT using targeted verteporfin with 4.5 mg/m2 and laser fluences of 10 (arrowhead), 25 (arrows), or 50 (asterisk) J/cm2. D, Late-phase fluorescein angiogram shows no hyperfluorescence or leakage from CNV consistent with CNV closure at the treatment spots using 10 (arrowhead) or 25 (arrows) J/cm2. Hyperfluorescence is noted at the edge of a treatment spot treated with 50 J/cm2 (asterisk).
|
|
|
Effective CNV closure was demonstrated by fluorescein angiography with targeted verteporfin and verteporfin-PVA at all tested photosensitizer doses. Table 1 and the histogram (Figure 4) summarize the effect of PDT on CNV, using different photosensitizer and light doses. Although the total number of treated CNVs was small, 100% closure of CNV was achieved using targeted verteporfin with drug doses as low as 3 mg/m2 and a light dose of 10 J/cm2. Similarly, for verteporfin-PVA, 100% closure was achieved using 3 mg/m2 and a light dose of 25 J/cm2.
|
|
|
|
Angiographic Grading of CNV Closure 24 Hours After PDT*
|
|
|
|
|
|
|
Figure 4. Histogram summarizes the effect of photodynamic therapy on choroidal neovascularization (CNV) using different photosensitizers and light doses as graded angiographically. All of the drug and light variables tested for each formulation achieved closure of CNV at some percentage. VEGFR-2–targeted verteporfin indicates verteporfin targeted to CNV by binding to vascular endothelial growth factor receptor-2; verteporfin-PVA, verteporfin conjugated to a modified polyvinyl alcohol polymer.
|
|
|
HISTOPATHOLOGIC FINDINGS IN TREATED CNV
All lesions defined as closed, regardless of whether targeted verteporfin or verteporfin-PVA was used and regardless of the light energy dose, shared similar histological features. Figure 5 shows a section of a CNV membrane treated with 4.5 mg/m2 of targeted verteporfin and 10 J/cm2 at 24 hours after PDT. This lesion was graded angiographically as closed. Vessels within the CNV showed vacuolization of endothelial cells and occlusion with platelets, fibrin, and erythrocytes. Extravasated erythrocytes were noted, and macrophages were seen within and around the treated CNV complex. Proliferating RPE cells can also be seen surrounding the CNV complex. Gross disruption of the outer retina, RPE, and Bruch membrane was generally attributed to the laser injury inducing the CNV.
|
|
|
|
Figure 5. Light micrographs of choroidal neovascularization 24 hours after photodynamic therapy using verteporfin conjugated to a modified polyvinyl alcohol polymer (A) and targeted verteporfin (B). The treatment variables included a drug dose of 4.5 mg/m2 and a laser fluence of 10 J/cm2. Capillaries (arrowheads) within the lesion are closed (toluidine blue, original magnification x40).
|
|
|
SELECTIVITY: HISTOLOGICAL GRADING OF PDT ON NORMAL CHOROID AND RETINA
Treatment selectivity was investigated by performing PDT in normal retina and choroid, using a qualitative assessment of angiographic findings after PDT and the histological grading scheme previously described.6 Photodynamic therapy for normal retina and choroid using verteporfin-PVA at a dose of 2.5 or 4.5 mg/m2 and irradiation 1 hour after drug injection using a fluence of 25 or 50 J/cm2 gave similar results. Fluorescein angiography 24 hours after PDT showed hypofluorescence in the area of treatment in the early phase of the angiogram with late hyperfluorescence (Figure 6A and B). Histological examination revealed occlusion of the choriocapillaris with RPE necrosis and pyknosis of the ONL ranging from occasional to less than 10%, with mild vacuolization and disarray of the inner and outer segments (Figure 7). The inner retinal layers and larger choroidal vessels, however, showed no damage, and the lesions were classified as grade 1 damage according to the published scheme.
|
|
|
|
Figure 6. A and B, Fluorescein angiography of normal choroid 24 hours after photodynamic therapy (PDT) using verteporfin conjugated to a modified polyvinyl alcohol polymer (4.5 mg/m2) and laser fluence of 25 (arrowhead) or 50 (empty arrowhead) J/cm2 showing early hypofluorescence and mild hyperfluorescence in the late frame (B). Marker laser spots are indicated with arrows. C and D, Fluorescein angiogram of normal choroid 24 hours after PDT treated with targeted verteporfin (4.5 mg/m2) and laser fluence of 25 J/cm2. No hyperfluorescence is noted in the treated area (arrowhead). Marker laser spots (arrows) were used to ensure identification of the treated area.
|
|
|
|
|
|
|
Figure 7. Light microscopy of normal choroid 24 hours after photodynamic therapy using verteporfin conjugated to a modified polyvinyl alcohol polymer (4.5 mg/m2) and laser fluence of 50 J/cm2. The choriocapillaris (cc) was occluded by thrombus, and the retinal pigment epithelium was necrotic. Vacuolization and disarray of the inner and outer segments (OS) were seen. Pyknosis of the photoreceptor nuclei (outer nuclear layer [ONL]) was less than 10%, and the inner retina appeared intact. This lesion was classified as having grade 1 damage. Arrow marks the level of the Bruch membrane (toluidine blue, original magnification x40).
|
|
|
Photodynamic therapy for normal retina and choroid using targeted verteporfin at a dose of 3 or 4.5 mg/m2 and irradiation 1 hour after drug injection using a fluence of 10, 25, or 50 J/cm2 showed even milder effects than the PDT using verteporfin-PVA. Fluorescein angiography performed 24 hours after PDT for normal retina and choroid using targeted verteporfin with light applied at 1 hour after photosensitizer injection showed no change in the area of treatment (Figure 6C and D). Lesions were difficult to find by light microscopy, and marker lesions were used to ensure localization. The retina appeared normal in almost all respects, with all retinal layers appearing similar to control areas at all doses of targeted verteporfin and all light fluences with negligible effect on RPE. A few pyknotic nuclei were seen in the ONL at all tested doses, always less than 5%, and usually only a few per field (Figure 8). All lesions were classified as grade 1, but showed much less damage than the typical grade 1 lesion because there was minimal effect on the RPE and virtually none on the photoreceptors. Closure of the choriocapillaris was the single consistent marker of PDT damage. Electron microscopy (Figure 9) confirmed the occlusion of the choriocapillaris by platelets, leukocytes, erythrocytes, and occasionally clumps of fibrin. The choriocapillaris endothelium was usually damaged and often missing entirely. No extravasation of cells or fibrin was seen. This PDT effect contrasted with the condition of the RPE adjacent to the closed choriocapillaris. No necrotic RPE cells were seen; most RPE cells had normal-appearing mitochondria and intact basal infoldings. Intracellular vacuoles were occasionally seen. Rare pyknosis was observed in the ONL, but the inner segment mitochondria appeared normal. The outer segments showed some disorganization but not much vacuolization. No changes were seen in the cells and capillaries in the inner retina.
|
|
|
|
Figure 8. Light microscopy of normal choroid 24 hours after photodynamic therapy using targeted verteporfin (4.5 mg/m2) and laser fluence of 50 J/cm2. A, The retinal layers appear to be well preserved. Occasional pyknotic nuclei are seen in the outer nuclear layer (ONL) as indicated by arrowheads (original magnification x40). The Bruch membrane is marked by the arrow. The lesion was classified as grade 1 damage. B, There is closure of choriocapillaris (cc) with well-preserved overlying layer of retinal pigment epithelium (RPE). OS indicates outer segments (original magnification x100).
|
|
|
|
|
|
|
Figure 9. Electron microscopy of normal choroid 24 hours after photodynamic therapy using targeted verteporfin (4.5 mg/m2) and laser fluence of 50 J/cm2. There is closure of choriocapillaris with red blood cells (r), platelets (p), and fibrin in the lumen. The endothelium is missing (arrow). Mitochondria (m) in the retinal pigment epithelium appear normal, and well-preserved basal infoldings remain (asterisk). Bar indicates 1 µm.
|
|
|
COMMENT
Photodynamic therapy with verteporfin as currently practiced has relative selectivity for CNV based on specific treatment variables, including drug, light dose, and timing of light application.1-2,5-6,30 However, a wealth of experimental data in tumor animal models indicates that small molecules, which do not bind specifically to a tumor marker, discriminate poorly between tumor and normal tissues in vivo.31-32 Similarly, preclinical studies have shown that PDT using untargeted verteporfin for CNV and normal retina and choroid causes some damage to normal structures. The use of homing vehicles, such as a recombinant monoclonal antibody or a targeting peptide to VEGFR-2 with the ability to selectively target neovascular endothelium, should be useful in improving PDT outcomes and expanding its applications.
Our choice of a peptide as a homing vehicle was multifactorial and included the ease of synthesis and modification, the lack of tissue cross-reactivity, a minimized immunological reaction, a low cost of production, and, most important, the potential incorporation of multiple targeting peptides to the photosensitizer-carrier complex. This last factor might allow one to target different molecular markers expressed by the pathological tissue to achieve even higher levels of specificity by directing the cytotoxic agent through a distinct routing path to the desired cell or subcellular compartment.33-36
Angiography demonstrated peak localization of the targeted verteporfin and verteporfin-PVA to CNV by 1 hour after intravenous administration, with both drugs clearing from the CNV by 2 hours. In contrast, liposomal verteporfin reaches peak intensity in rat CNV at 15 to 20 minutes after intravenous administration and clears within 30 minutes.28 Targeted verteporfin and verteporfin-PVA are relatively large molecules (28-30 kDa compared with 718 Da for unbound verteporfin), and this probably accounts for the later and more prolonged localization to CNV. To lentino and colleagues37 demonstrated localization of fluoresceinated dextrans and antibodies in experimental CNV after intravenous administration and a correlation with molecular weight and radius. Presumably, molecules of this size are able to exit the vascular space through fenestrations in the choriocapillaris and CNV, but are less readily cleared than are smaller molecules. Thus it is not surprising that PDT using both larger molecules, targeted verteporfin and verteporfin-PVA, showed greater efficacy for CNV closure with lower drug and light doses than those seen with PDT using unbound verteporfin. One hundred percent closure was achieved with as little as 3 mg/m2 and 10 J/cm2 for targeted verteporfin and 25 J/cm2 for verteporfin-PVA, although even with 3 or 6 mg/m2 and 25 J/cm2, the closure rate ranges from 83% to 92%.28
Although the efficacy of CNV closure was similar for PDT using targeted verteporfin and verteporfin-PVA, the drugs differed somewhat in their selectivity. Angiography after PDT of normal retina and choroid using verteporfin-PVA demonstrated early hypofluorescence and late leakage 24 hours after PDT, and results of the histological examination showed grade 1 lesions with RPE necrosis, mild pyknosis of the photoreceptor nuclei, and vacuolization and disarray of the inner and outer segments. Although this damage is still mild, it is no better than PDT with standard verteporfin. One can speculate that the verteporfin-PVA can still leak through the CNV without binding to the endothelium, reaching the extravascular space and clearing slowly. In contrast, PDT for normal retina and choroid using targeted verteporfin did not show any angiographic hypofluorescence or late leakage 24 hours after PDT, and results of the histological examination showed minimal effect on the RPE and no injury to photoreceptors. Although these lesions were formally classified as grade 1 lesions, the observed damage was substantially less. Presumably, the VEGFR-2–targeted verteporfin localizes to CNV on the basis of size in a manner similar to verteporfin-PVA, but can then bind VEGFR-2 receptors expressed on neovascular endothelium. This binding could lead to increased efficacy of CNV closure and increased selectivity, because the photosensitizer would be sequestered at the endothelium and would spare the RPE and photoreceptors.
An additional advantage to PDT using VEGFR-2–targeted verteporfin might be the combination of an antiangiogenic effect with PDT. Previous studies from our group9-10 have shown that combining antiangiogenic agents with PDT causes a selective increased cytotoxicity to neovascular endothelial cells in vitro and in vivo. The VEGFR-2–targeting peptide used in the present study has been shown by Binetruy-Tournaire and colleagues21 to completely inhibit VEGF-induced angiogenesis. Thus, VEGFR-2–targeted verteporfin has the potential to exert an antiangiogenic potentiating effect before its photoactivation.
CONCLUSIONS
We showed that the efficacy of PDT can be enhanced by conjugating the photosensitizer to PVA. In addition, selectivity of PDT can be enhanced by targeting the photosensitizer to VEGFR-2. If these preliminary findings are substantiated in primate models of CNV, clinical studies may be warranted to determine whether vision outcomes can be improved. Our results also highlight the utility of designing peptide photosensitizer conjugates as vehicles for regulating the distribution of photosensitizer to CNV to maximize their selectivity in PDT. In the future, other candidate homing molecules may be identified with even greater specificity for neovascular endothelium. These preliminary results indicate that targeted PDT for CNV is feasible and may offer a qualitative improvement in current clinical therapies.
AUTHOR INFORMATION
| |
Correspondence: Joan W. Miller, MD, Angiogenesis and Laser Research Laboratory, Retina Service, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114 (jwmiller{at}meei.harvard.edu).
Submitted for publication August 12, 2002; final revision received October 23, 2003; accepted January 30, 2004.
This study was supported by the Foundation Fighting Blindness, Owings Mills, Md (Drs Renno, Terada, and Miller), and the Iacocca Foundation, Boston, Mass (Dr Renno).
From the Angiogenesis and Laser Laboratory, Retina Service, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston (Drs Renno, Terada, Gragoudas, and Miller and Mr Michaud), and the Department of Chemistry, American University of Beirut, Beirut, Lebanon (Dr Haddadin). Dr Renno is now with the Department of Ophthalmology, Jules Stein Eye Institute, University of California–Los Angeles. The Massachusetts Eye and Ear Infirmary has an ownership interest in 3 US patents directed to the use of verteporfin. In addition, the Massachusetts Eye and Ear Infirmary has an ownership interest in certain patent applications directed to the selective destruction of subretinal choroidal neovasculature for the treatment of macular degeneration and other disorders. Should the Massachusetts Eye and Ear Infirmary receive royalties or other financial remuneration as a result of these patents and patent applications, Drs Miller, Renno, and Gragoudas would receive a share of the same in accordance with the Massachusetts Eye and Ear Infirmary's institutional patent policy and procedures, which includes royalty-sharing provisions.
REFERENCES
1. Miller J, Schmidt-Erfurth U, Sickenberg M, et al. Photodynamic therapy for choroidal neovascularization due to age-related macular degeneration with verteporfin: results of a single treatment in a phase 1 and 2 study. Arch Ophthalmol. 1999;117:1161-1173. [published correction appears in Arch Ophthalmol. 2000;118:488]
FREE FULL TEXT
2. Treatment of Age-Related Macular Degeneration With Photodynamic Therapy (TAP) Study Group. Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with verteporfin: one-year results of 2 randomized clinical trials: TAP report 1. Arch Ophthalmol. 1999;117:1329-1345. [published correction appears in Arch Ophthalmol. 2000;118:488]
FREE FULL TEXT
3. Bressler N, Treatment of Age-Related Macular Degeneration With Photodynamic Therapy (TAP) Study Group. Photodynamic therapy of subfoveal choroidal neovascularization in related age macular degeneration with verteporfin: two-year results of randomized clinical trials: TAP report 2. Arch Ophthalmol. 2001;119:198-207.
FREE FULL TEXT
4. Sickenberg M, Schmidt-Erfurth U, Miller J, et al. A preliminary study of photodynamic therapy using verteporfin for choroidal neovascularization in pathologic myopia, ocular histoplasmosis syndrome, angioid streaks, and idiopathic causes. Arch Ophthalmol. 2000;118:327-336.
FREE FULL TEXT
5. Miller J, Walsh A, Kramer M, et al. Photodynamic therapy of experimental choroidal neovascularization using lipoprotein-delivered benzoporphyrin. Arch Ophthalmol. 1995;113:810-818.
FREE FULL TEXT
6. Kramer M, Miller J, Mihaud N, et al. Liposomal benzoporphyrin derivative verteporfin photodynamic therapy. Ophthalmology. 1996;103:427-438.
ISI
| PUBMED
7. Husain D, Kramer M, Kenney A, et al. Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to seven weeks after treatment. Invest Ophthalmol Vis Sci. 1999;40:2322-2331.
FREE FULL TEXT
8. Reinke M, Canakis C, Hussain D, et al. Verteporfin photodynamic therapy (PDT) retreatment of normal retina and choroid in the cynomolgus monkey. Ophthalmology. 1999;106:1915-1923.
FULL TEXT
|
ISI
| PUBMED
9. Renno R, Delori F, Holzer R, Gragoudas E, Miller J. Photodynamic therapy using Lu-Tex induces apoptosis in vitro and shows potentiate action combined with angiostatin in retinal capillary endothelial cells. Invest Ophthalmol Vis Sci. 2000;41:3963-3971.
FREE FULL TEXT
10. Gauthier D, Husain D, Kim I, et al. Safety and Efficacy of Intravitreal Injection of rhuFab VEGF in Combination With Verteporfin PDT on Experimental Choroidal Neovascularization. Fort Lauderdale, Fla: Association for Research in Vision & Ophthalmology; 2002.
11. Pasqualini R, Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature. 1996;380:364-366.
FULL TEXT
| PUBMED
12. Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science. 1998;279:377-380.
FREE FULL TEXT
13. Arap WHW, Bernasconi M, Kain R, et al. Targeting the prostate for destruction through a vascular address. Proc Natl Acad Sci U S A. 2002;99:1527-1531.
FREE FULL TEXT
14. Miller J, Adamis A, Shima D, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol. 1994;145:574-584.
ABSTRACT
15. Aiello L, Pierce E, Foley E, et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci U S A. 1995;92:10457-10461.
FREE FULL TEXT
16. McLeod D, Taomoto M, Cao J, Zhu Z, Witte L, Lutty G. Localization of VEGF receptor-2 (KDR/Flk-1) and effects of blocking it in oxygen-induced retinopathy. Invest Ophthalmol Vis Sci. 2002;43:474-482.
FREE FULL TEXT
17. Tille J, Wood J, Mandriota S, et al. Vascular endothelial growth factor (VEGF) receptor-2 antagonists inhibit VEGF- and basic fibroblast growth factor–induced angiogenesis in vivo and in vitro. J Pharmacol Exp Ther. 2001;299:1073-1085.
FREE FULL TEXT
18. Krzystolik M, Afshari M, Adamis A, et al. Prevention of experimental choroidal neovascularization with intravitreal anti–vascular endothelial growth factor antibody fragment. Arch Ophthalmol. 2002;120:338-346.
FREE FULL TEXT
19. Kim I, Ryan A, Rohan R, et al. Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes. Invest Ophthalmol Vis Sci. 1999;40:2115-2121.
FREE FULL TEXT
20. Wada M, Ogata N, Otsuji T, Uyama M. Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization. Curr Eye Res. 1999;18:203-213.
FULL TEXT
|
ISI
| PUBMED
21. Binetruy-Tournaire R, Demangel C, Malavaud B, et al. Identification of a peptide blocking vascular endothelial growth factor (VEGF)–mediated angiogenesis. EMBO J. 2000;19:1525-1533.
FULL TEXT
|
ISI
| PUBMED
22. Lange N, Ballini J, Wagnieres G, Bergh HVD. A new drug-screening procedure for photosensitizing agents used in photodynamic therapy for CNV. Invest Ophthalmol Vis Sci. 2001;42:38-46.
FREE FULL TEXT
23. Steele K, Liu D, Davis N, Deal H, Levy J. The preparation and application of porphyrin-monocalonal antibodies for cancer therapy. In: Dougherty TJ, ed. Photodynamic Therapy: Mechanisms: 19-20 January 1989, Los Angeles, California. Vol 1065. Bellingham, Wash: International Society for Optical Engineering; 1989:73-79.
24. Jiang FN, Jiang S, Liu D, Richter A, Levy JG. Development of technology for linking photosensitizers to a model monoclonal antibody. J Immunol Methods. 1990;134:139-149.
FULL TEXT
|
ISI
| PUBMED
25. Monner D. An assay for growth of mouse bone marrow cells in microtiter liquid culture using the tetrazolium salt MTT, and its application to studies of myeloporesis. Immunol Lett. 1988;19:261-268.
FULL TEXT
|
ISI
| PUBMED
26. Dobi E, Puliafito C, Destro M. A new model of subretinal neovascularization in the pigmented rat. Arch Ophthalmol. 1989;107:264-269.
FREE FULL TEXT
27. Tobe T, Takahashi K, Ohkuma H, Uyamam M. Experimental choroidal neovascularization in the rat [in Japanese]. Nippon Ganka Gakkai Zasshi. 1994;98:837-845.
PUBMED
28. Zacks D, Ezra E, Terada Y, et al. Verteporfin photodynamic therapy in the rat model of choroidal neovascularization: angiographic and histologic characterization. Invest Ophthalmol Vis Sci. 2002;43:2384-2391.
FREE FULL TEXT
29. Gilpin D. Calculation of a new Meeh constant and experimental determination of burn size. Burns. 1996;22:607-611.
FULL TEXT
|
ISI
| PUBMED
30. Husain D, Miller J, Michaud N, Connolly E, Flotte T, Gragoudas E. Intravenous infusion of liposomal benzoporphyrin derivative for photodynamic therapy of experimental choroidal neovascularization. Arch Ophthalmol. 1996;114:978-985.
FREE FULL TEXT
31. Murdter TE, Sperker B, Kivisto KT, et al. Enhanced uptake of doxorubicin into bronchial carcinoma: beta-glucuronidase mediates release of doxorubicin from a glucuronide prodrug (HMR 1826) at the tumor site. Cancer Res. 1997;57:2440-2445.
FREE FULL TEXT
32. Folli S, Westermann P, Braichotte D, et al. Antibody-indocyanin conjugates for immunophotodetection of human squamous cell carcinoma in nude mice. Cancer Res. 1994;54:2643-2649.
FREE FULL TEXT
33. Vocero-Akbani AM, Heyden NV, Lissy NA, Ratner L, Dowdy SF. Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. Nat Med. 1999;5:29-33.
FULL TEXT
|
ISI
| PUBMED
34. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 1999;285:1569-1572.
35. Schwarze SR, Dowdy SF. In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci. 2000;21:45-48.
36. Lindgren M, Hallbrink M, Prochiantz A, Langel U. Cell-penetrating peptides. Trends Pharmacol Sci. 2000;21:99-103.
FULL TEXT
| PUBMED
37. Tolentino M, Husain D, Theodosiadis P, et al. Angiography of fluoresceinated anti-vascular endothelial growth factor antibody and dextrans in experimental choroidal neovascularization. Arch Ophthalmol. 2000;118:78-84.
FREE FULL TEXT
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati
What's this?
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES
Photoreceptor Protection after Photodynamic Therapy Using Dexamethasone in a Rat Model of Choroidal Neovascularization
She et al.
IOVS 2008;49:5008-5014.
ABSTRACT
| FULL TEXT
Drug Delivery to Posterior Intraocular Tissues: Third Annual ARVO/Pfizer Ophthalmics Research Institute Conference
Edelhauser et al.
IOVS 2008;49:4712-4720.
FULL TEXT
Reduced Photoreceptor Damage after Photodynamic Therapy through Blockade of Nitric Oxide Synthase in a Model of Choroidal Neovascularization
She et al.
IOVS 2007;48:2268-2277.
ABSTRACT
| FULL TEXT
Metabolic Profile of a Peptide-Conjugated Chlorin-Type Photosensitizer Targeting Neuropilin-1: An in Vivo and in Vitro Study
Tirand et al.
Drug Metab. Dispos. 2007;35:806-813.
ABSTRACT
| FULL TEXT
The Nf2 tumor suppressor regulates cell-cell adhesion during tissue fusion
McLaughlin et al.
Proc. Natl. Acad. Sci. USA 2007;104:3261-3266.
ABSTRACT
| FULL TEXT
|
