Chloroaluminum Sulfonated Phthalocyanine Research Articles

201 POSTER Dolichyl phosphate cycle stimulation as a possible mechanism of mdr1 gene suppression in p-388 leukemia cells

Purpose: Photodynamic therapy (PDT) has been demonstrated to inhibit experimental intimal hyperplasia and to lead to expedient reendothelialization but negligible repopulation of the vessel media. The mechanism that underlies the differential ingrowth of cells into PDT-treated vessel segments is not undertood. Because the extracellular matrix (ECM) is known to modulate specific cell functions, this study was designed to determine whether PDT of isolated ECM affects the function of endothelial cells (ECs) and smooth muscle cells (SMCs).Methods: PDT of bovine aortic EC-ECM was performed with chloroaluminum sulfonated phthalocyanine and 675-nm laser light. Control specimens included untreated ECM, ECM-free plates, and ECM exposed to either light or photosensitizer only. Cell function was characterized by attachment, proliferation, and migration of ECs or SMCs that were plated onto identically treated matrixes.Results: SMC attachment (86%±0.4% vs 95%±0.4%), proliferation (46%±0.5% vs 100%±1.4%), and migration (40%±1.0% vs 100%±0.9%) were significantly inhibited after PDT of ECM when compared with untreated ECM (all p<0.001). In contrast, PDT and migration (118%±1.2% vs 100%±0.8; p<0.01), but did not affect attachment.Conclusions: This report establishes PDT-induced changes in the ECM with a result of inhibition of SMCs and stimulation of EC functions. It provides insight into how PDT-treated arteries can develop favorable EC repopulation without SMC-derived intimal hyperplasia. These findings may help provide a better understanding of the interactions between cells and their immediate environment in vascular remodeling.

Photodynamic Therapy Induces Apoptosis in Intimal Hyperplastic Arteries

Photodynamic therapy (PDT) generates free radicals through the absorption of light by photosensitizers. PDT shows promise in the treatment of intimal hyperplasia, which contributes to restenosis, by completely eradicating cells in the vessel wall. This study investigates the mechanisms of PDT-induced cell death. PDT, using the photosensitizer chloroaluminum-sulfonated phthalocyanine (1 mg/kg) and laser light (lambda = 675 nm) 100 J/cm(2) was administered to rat carotid arteries after balloon injury-induced intimal hyperplasia. Apoptosis was determined by cell morphology with light microscopy and transmission electron microscopy, DNA cleavage by terminal dUTP nick-end labeling staining, and nucleosomal fragmentation (ladder pattern) by DNA agarose gel electrophoresis. Four hours after PDT, apoptosis was observed in vascular cells, as evidenced by terminal dUTP nick-end labeling staining and transmission electron microscopy. Within 24 hours no cells were present in the neointima and media. Immunofluorescence using an alpha-smooth muscle cell actin antibody confirmed the disappearance of all neointimal and medial cells within 24 hours. No inflammatory cell infiltrate was observed during this time frame. Apoptosis was sharply confined to the PDT treatment field. These data demonstrate that vascular PDT induces apoptosis as a mechanism of rapid, complete, and precise cell eradication in the artery wall. These findings and the lack of inflammatory reaction provide the basis for understanding and developing PDT for a successful clinical application in the treatment of hyperplastic conditions such as restenosis.

Photodynamic therapy generates a matrix barrier to invasive vascular cell migration.

Photodynamic therapy (PDT) inhibits experimental intimal hyperplasia. PDT results in complete vascular wall cell eradication with subsequent adventitia but minimal media repopulation. This study was designed to test the hypothesis that PDT alters the vascular wall matrix thereby inhibiting invasive cell migration, and as such, provides an important barrier mechanism to favorably alter the vascular injury response. Untreated smooth muscle cells (SMCs) and fibroblasts were seeded on control and PDT-treated (100 J/cm(2); photosensitizer was chloroaluminum-sulfonated phthalocyanine, 5 microg/mL) 3-dimensional collagen matrix gels. Invasive cell migration was temporally quantified by calibrated microscopy. Zymography and ELISA assessed SMC matrix metalloproteinase levels. Molecular changes of gel proteins and their susceptibility to collagenase were analyzed by SDS-PAGE and Western blot. Limited pepsin digestion and histology were used to assess the in vivo relevance of the model, using an established rat carotid artery model at 1 and 4 weeks after balloon injury and PDT. PDT of 3-dimensional matrix of gels led to a 52% reduction of invasive SMCs and to a 59% reduction of fibroblast migration (P<0.001) but did not significantly affect secretion of matrix metalloproteinases. PDT induced collagen matrix changes, including cross-linking, which resulted in resistance to protease digestion. PDT led to a durable 45% reduction in pepsin digestion susceptibility of treated arteries (P<0.001) and inhibition of periadventitial cell migration into the media. These data suggest that PDT of matrix gels generates a barrier to invasive cellular migration. This newly identified effect on matrix proteins underscores its pleiotropic actions on the vessel wall, and as such, PDT may be of considerable potential therapeutic value to inhibit restenosis.

Differential modulation of vascular endothelial and smooth muscle cell function by photodynamic therapy of extracellular matrix: Novel insights into radical-mediated prevention of intimal hyperplasia

Photodynamic therapy (PDT) has been demonstrated to inhibit experimental intimal hyperplasia and to lead to expedient reendothelialization but negligible repopulation of the vessel media. The mechanism that underlies the differential ingrowth of cells into PDT-treated vessel segments is not understood. Because the extracellular matrix (ECM) is known to modulate specific cell functions, this study was designed to determine whether PDT of isolated ECM affects the function of endothelial cells (ECs) and smooth muscle cells (SMCs). PDT of bovine aortic EC-ECM was performed with chloroaluminum sulfonated phthalocyanine and 675-nm laser light. Control specimens included untreated ECM, ECM-free plates, and ECM exposed to either light or photosensitizer only. Cell function was characterized by attachment, proliferation, and migration of ECs or SMCs that were plated onto identically treated matrixes. SMC attachment (86% +/- 0.4% vs 95% +/- 0.4%), proliferation (46% +/- 0.5% vs 100% +/- 1.4%), and migration (40% +/- 1.0% vs 100% +/- 0.9%) were significantly inhibited after PDT of ECM when compared with untreated ECM (all p < 0.001). In contrast, PDT of ECM significantly enhanced EC proliferation (129% +/- 6.2% vs 100% +/- 6.2%; p < 0.03) and migration (118% +/- 2% vs 100% +/- 0.8; p < 0.01), but did not affect attachment. This report establishes PDT-induced changes in the ECM with a result of inhibition of SMCs and stimulation of EC functions. It provides insight into how PDT-treated arteries can develop favorable EC repopulation without SMC-derived intimal hyperplasia. These findings may help provide a better understanding of the interactions between cells and their immediate environment in vascular remodeling.

Photothrombosis of retinal and choroidal vessels in rabbit eyes using chloroaluminum sulfonated phthalocyanine and a diode laser.

Photothrombosis is a relatively new photodynamic application leading to vascular occlusion. In the current work the effectiveness of phthalocyanine and a diode laser in photothrombosis of normal retinal and choroidal vessels was evaluated. Big retinal vessels of temporal myelin wing were irradiated using a 670 nm diode laser (2 mW, 0.5 mm2) after the injection of chloroaluminum sulfonated phthalocyanine (5 mg/kg) in twenty albino rabbits. Animals were followed up to a maximum of 7 months using fundus photography, fluoroangiography, and histology. Photothrombosis of the irradiated retinal vessels and of underlying choroidal vessels resulted in all treated eyes after 13 to 17.5 min of irradiation. The retinal vessels were patent again by the 7th day after the procedure. Choroidal vessels remained closed during the whole follow-up period. Light and electron microscopy demonstrated occupation of irradiated choroidal and retinal vessels by platelet thrombi. Damage of endothelial cell structure of these vessels could be seen. Outer retinal and RPE damage localized at irradiation area was observed. The combination of phthalocyanine with a low power diode laser is a simple and effective way for the induction of photodynamic thrombosis in fundus vessels.

Photothrombosis of retinal and choroidal vessels in rabbit eyes using chloroaluminum sulfonated phthalocyanine and a diode laser

Background and Objectives Photothrombosis is a relatively new photodynamic application leading to vascular occlusion. In the current work the effectiveness of phthalocyanine and a diode laser in photothrombosis of normal retinal and choroidal vessels was evaluated. Study design, Materials and Methods: Big retinal vessels of temporal myelin wing were irradiated using a 670 nm diode laser (2 mW, 0.5 mm2) after the injection of chloroaluminum sulfonated phthalocyanine (5 mg/kg) in twenty albino rabbits. Animals were followed up to a maximum of 7 months using fundus photography, fluoroangiography, and histology. Results Photothrombosis of the irradiated retinal vessels and of underlying choroidal vessels resulted in all treated eyes after 13 to 17.5 min of irradiation. The retinal vessels were patent again by the 7th day after the procedure. Choroidal vessels remained closed during the whole follow-up period. Light and electron microscopy demonstrated occupation of irradiated choroidal and retinal vessels by platelet thrombi. Damage of endothelial cell structure of these vessels could be seen. Outer retinal and RPE damage localized at irradiation area was observed. Conclusion The combination of phthalocyanine with a low power diode laser is a simple and effective way for the induction of photodynamic thrombosis in fundus vessels. © 1996 Wiley-Liss, Inc.

Long term follow-up of retinal and choroidal structures after experimental vascular thrombosis using chloroaluminum sulfonated phthalocyanine and a diode laser

Long term follow-up of retinal and choroidal structures after experimental vascular thrombosis using chloroaluminum sulfonated phthalocyanine and a diode laser

Photodynamic therapy of vein grafts: suppression of intimal hyperplasia of the vein graft but not the anastomosis.

There is no clinically useful therapy for the suppression of vein bypass graft intimal hyperplasia (IH). Photodynamic therapy (PDT), a technique that uses light to activate otherwise biologically inert photosensitizers to produce cytotoxic effects, has been demonstrated to successfully inhibit experimental IH in balloon-injured arteries. The purpose of this study was to investigate the efficacy of PDT as a method to reduce vein graft IH. Reversed external jugular vein bypass grafts of the common carotid artery were performed in 28 male Sprague-Dawley rats. The animals received either chloroaluminum sulfonated phthalocyanine (2.5 mg/kg intravenously) 24 hours before the ex vivo irradiation of the vein grafts (VG) with 100 joule/cm2 at 675 nm (PDT VG) or saline solution as control (CON VG). Preharvest bromodeoxyuridine was administered to label proliferating cells. All vein grafts were perfusion fixed within 96 hours for a pilot study or at 2 and 4 weeks for the main study. Histology, immunohistochemistry, and morphometric analysis were performed. There was no acute thrombus formation in the hypocellular PDT VG with occasional platelets but no leukocytes adherent to the luminal surface. Intimal areas of the PDT VG were 18% at 2 weeks and 53% at 4 weeks of the CON VGs (p < 0.05). Medial areas and percent of stenoses were also significantly less in PDT than in CON VG. However, intimal hyperplasia noted in the longitudinal sections within 2 mm of the anastomoses did not demonstrate a difference between PDT and CON VG. Intimal hyperplasia of both PDT and CON VG consisted of smooth muscle cells, verified by immunohistochemistry. Bromodeoxyuridine-labeled cells were more abundant in 2-week than in 4-week specimens, were found most frequently in the intimal areas of the CON VG body, and were equivalent in the anastomoses of PDT VG and CON VG. These data suggest that PDT of vein grafts suppresses the development of IH in the body of the vein graft but does not affect IH adjacent to the anastomoses. The artery may be the source of proliferating smooth muscle cells that contribute to the anastomotic vein graft IH.

Variation in the distribution of a phthalocyanine photosensitizer in naturally occurring tumors of animals

Jcmllldd J J AND PIIOII:il]I:IIK~ Journal of P h o t o c h e m i s t r y and Photobiology n , ~ B: Biology 27 (1995) 271-277 ELSEVIER News and Views Variation in the distribution of a phthalocyanine photosensitizer in naturally occurring tumors of animals George M. Peary, Tatiana B. Krasieva, Bruce J. Tromberg, E. Dave Eusantos, Michael W. Berns Beckman Laser Institute and Medical Clinic, College of Medicine, University of California, Irvine, CA 92715, USA 1. Introduction The biodistribution and cytotoxicity of photosensi- tizers have been shown to vary in tumors, not only between classes of photosensitizer [1-5], but also be- tween differently sulfonated forms of the same parent compound [6-14]. For example, the distribution has been reported to be different for related phthalocyanine compounds when viewed in the same tumor model [14,15]. In addition, the local hypoxia which results from early vascular collapse induced by photodynamic therapy (PDT) has been observed to protect tumor cells from the direct effects of photodynamic cytotoxicity in vivo by limiting the oxygen availability for singlet oxygen generation [16], further suggesting that the distribution of a photosensitizer within a tumor can influence its efficiency for that tumor type. It has been proposed that a variety of tissue factors may be responsible for the preferential uptake and/or retention of hematoporphyrin derivative [17]. It is con- ceivable that these factors could vary substantially be- tween tumor types. When different tumors were im- planted on the chick chorioallantoic membrane (CAM) model, photosensitizer uptake varied with tumor type [18]. Thus the uptake and distribution of a photosen- sitizer are clearly complex functions which may be influenced by both the chemical properties of the pho- tosensitizer and the physiologic behavior of a specific tumor which may vary between tumor types. In this report, we evaluate the intratumor distribution of a phthalocyanine preparation in three different, naturally occurring, domestic dog and cat tumors. Our goal was to determine whether the same intratumor biodistribution could be observed in a variety of naturally occurring tumors. Lack of uniformity would highlight 1011-1344/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 1011-1344(94)07075-X problems in making generalizations or applying infor- mation about tumor biodistribution, mechanisms of action or efficiency of a specific photosensitizer based on a limited tumor model. We conclude that it is important to study the photosensitizer biodistribution and efficiency in naturally occurring tumor lines in order to determine whether differences exist between tumor types, and if such can be correlated to clinical response. 2. Experimental details A chloroaluminum sulfonated phthalocyanine (AISPc) preparation was used in clinical studies of the response of spontaneously occurring, natural tumors of domestic dogs and cats to PDT. The product was obtained in solution at a concentration of 300 mg ml- 1 (Ciba-Geigy Dyestuffs and Chemicals, Basel, Switzer- land), lyophilized to a powder, resuspended in Dul- becco's phosphate-buffered saline ( 1 × ) (GIBCO, Grand Island, NY) to a concentration of 1 mg ml-1, placed in 6 ml or 10 ml amber vials and stored frozen at - 2 0 °C, in the dark, until needed for patient treatment. High pressure liquid chromatography was used to identify and quantify the phthalocyanine content of the prepared solution, revealing that it is composed primarily of trisulfonated ($3) and tetrasulfonated ($4) compounds, with some disulfonated ($2) compound and a highly sulfonated A1Pc compound possibly containing a sulfonate as an axial ligand on the central metal ion. Veterinary patients received an intravenous injection of the AISPc preparation at a dosage of 1 mg kg-1 of body weight. After 48 h, samples of tumor tissue were obtained, placed in embedding medium for frozen tissue specimens (OCT Compound, Tissue-Tek-Miles, Elk-

Photodynamic laser cyclodestruction with chloroaluminum sulfonated Phthalocyanine (CASPc) or photofrin®(PII) Vs. Nd:YAG laser cyclodestruction in a pigmented rabbit model

To investigate Photofrin (PII) and CASPc for photodynamic therapy (PDT) of the ciliary body in rabbits. PII (10 mg/kg) or CASPc (1 mg/kg) was given by ear vein. Pharmacokinetics were studied in frozen sections by fluorescence microscopy (CCD camera based low light detection system with digital image processing) at 1 and 24 h (8 rabbits; 16 eyes). Laser light was delivered (argon pumped dye laser; 630 and 675 nm; 8 rabbits; 16 eyes) by contact fiberoptic. To compensate for iris attenuation, irradiance was 125 mW/cm2 (20, 40, 80, or 160 J/cm2). Controls (4 rabbits; 8 eyes) received laser light without photochemicals (OD) and for comparison, continuous wave Nd:YAG laser by fiberoptic (0.8-1.2J; OS). Localization studies showed intravascular distribution with some selective ciliary body distribution at 24 h (PII > CASPc). Rabbits treated with PII or CASPc exhibited variable amounts of gross ciliary body edema, infarction, and necrosis by 24-48 h. This response was not seen in PDT control tissues; damage was seen in the iris and ciliary body, with partial vacuolization of the pigment epithelium. PDT may offer a more selective approach to ciliary body destruction. A small but significant thermal effect was seen during PDT from melanin photon uptake with damage to iris and ciliary body. Thermal damage and potential interaction with ocular visual pigments may limit use of these photochemicals and wavelengths for PDT of the ciliary body.

Biaxial elastic properties of rat arteries in vivo: influence of vascular wall cells on anisotropy.

There is no consensus as to the degree of arterial anisotropy or to its relationship to vascular cell function. Given the relevance of the isotropic assumption in formulating elasticity models, reliable measures of biaxial displacements are needed. In this study, a video motion analyzer (VMA) was used to describe the biaxial in vivo dynamic elasticity of 22 carotid arteries and 5 abdominal aortas in 27 rats. The influence of vascular cell function was also examined by subjecting six rats to a photosensitive drug, chloroaluminum sulfonated phthalocyanine (CASPc), which is focally cytotoxic on activation by laser. Circumferential compliance (Ccirc) was greater than longitudinal compliance (Clong) for all vessels. Compliance pressure curves were nonlinear, and biaxial displacements were in phase. The circumferential elastic modulus was less than the longitudinal modulus at common stresses. CASPc + laser reduced Ccirc but not Clong, thus altering Poisson's ratio. In conclusion, rat arteries are biaxially, nonlinearly elastic and anisotropic in vivo. Vascular cells modulate Poisson's ratio by influencing Ccirc.

Phthalocyanine photodynamic therapy: new strategy for closure of choroidal neovascularization.

Chloro-aluminum sulfonated phthalocyanine (CASPc) is a photo-chemically active dye employed in photodynamic therapy (PDT). CASPc is a potent generator of singlet oxygen when irradiated with 675 nm light and is also capable of fluorescence, allowing visualization of the dye in tissues. We devised an angiography system using CASPc fluorescence to determine its localization in experimental choroidal neovascularization in monkeys and then investigated the ability of CASPc to produce photochemical closure of neovascularization upon irradiation with 675nm laser light. Fluorescent imaging indicated that CASPc localized angiographically in areas of neovascularization for at least 24 hours. Irradiation with 675 nm laser light 5-30 minutes after CASPc injection produced complete closure of choroidal neovascularization with minimal damage to overlying retina. We conclude that CASPc localizes in neovascular choroidal vessels and that CASPc photodynamic therapy can produce closure of these choroidal vessels.

Retinal and choroidal vessel closure using phthalocyanine photodynamic therapy.

Chloro-aluminum sulfonated phthalocyanine (CASPc) is a photoactive dye capable of generating photochemical reactions when excited with 675 nm light. We used CASPc to produce photochemical closure of retinal medullary ray vessels and choroidal vessels in normal rabbits. Irradiation prior to CASPc injection produced no photographic, angiographic, or histologic lesions in any eyes. Identical irradiation of medullary ray and choroidal vessels after CASPc injection produced complete vessel closure in all eyes. Histopathologic examination showed marked thrombosis of medullary ray and choroidal vessels, with minimal damage to contiguous tissues including the neurosensory retina. We conclude that CASPc can produce profound closure of normal retinal and choroidal vessels with minimal deleterious effect on surrounding tissues.

Phthalocyanine mediated photodynamic thrombosis of experimental corneal neovascularization: effect of phthalocyanine dose and irradiation onset time on vascular occlusion rate.

The aim of this study was to evaluate the possible influence of phthalocyanine dose and of time interval between phthalocyanine injection and irradiation commencement on the rate of experimental corneal neovascularization photodynamic thrombosis in albino rabbits. New corneal vessels were irradiated with a diode laser (670 nm, 2 mW) after the intravenous injection of chloroaluminum sulfonated phthalocyanine. Different animals were irradiated either 5 min after the injection of different phthalocyanine doses (3, 6, 8, 12, or 14 mg/kg), or at different times (5 min, 24 h, or 58 h) after a standard phthalocyanine dose (3 mg/kg) injection. Irradiation time necessary for vascular occlusion was recorded. Decrease of phthalocyanine dose as well as delay of irradiation onset resulted in a statistically significant increase of irradiation time. Electron and light histological examination revealed platelet thrombi inside irradiated corneal new vessels. Damage in the vascular endothelial cell membrane and in intercellular contact structure was noted, leading to disorganization of the endothelial cells layer and death of most endothelial cells. These results indicate that both early commencement of irradiation after phthalocyanine injection and phthalocyanine dose increase accelerate the rate of phthalocyanine mediated corneal neovascularization photodynamic thrombosis. Thrombosis seems to result from photochemically induced vascular endothelial cell damage.

Effectiveness of corneal neovascularization photothrombosis using phthalocyanine and a diode laser (675 nm)

We used chloroaluminum sulfonated phthalocyanine as a photo-sensitizer and a diode laser as a light source for induction of photothrombosis of corneal neovascularization. Corneal neovascularization was induced in 1 eye of each of 10 New Zealand white rabbits using intrastromal 6.0 silk sutures. After the intravenous injection of phthalocyanine in a dose of 4 mg per kg of body weight, photothrombosis was carried out using a diode laser emitting at 675 nm. The animals were followed up by both fluorescein angiography and slit-lamp photography for up to three months. At the end of the follow-up period the eyes were prepared for histology. After photothrombosis, thrombus formation was induced within new vessels demonstrating histological characteristics of both arterioles and venules. Most of the vessels disappeared or remained closed during the follow-up period. Recanalization of some of the thrombosed vessels occurred within the first 20 days after the operation. The combination of phthalocyanine and a 675 nm diode laser is effective for the induction of photothrombosis of corneal neovascularization.

Comparison of Intravenous and Intravesical Administration of Chloro-Aluminum Sulfonated Phthalocyanine for Photodynamic Treatment in a Rat Bladder Cancer Model

Photodynamic therapy is an experimental treatment of superficial bladder tumors. Photofrin, a mixture of porphyrins, is the only photosensitizer in clinical use in the U.S.A. and its major side effect is prolonged cutaneous phototoxicity. In order to circumvent this problem of phototoxicity, new photosensitizers are being examined. Cutaneous phototoxicity may also be minimized by local administration of photosensitizer. Therefore, in this study, we investigated the photosensitizer chloro-aluminum sulfonated phthalocyanine (CASPc) in vivo in a rat bladder carcinoma model, and compared two different routes of CASPc administration. AY-27 rat bladder carcinoma cells were transplanted into rat bladders. Eight days after tumor transplantation the biodistribution of CASPc in bladder, skin, muscle and bladder tumor was determined by fluorescence measurements after dye extraction. Photosensitizer administered by intravenous injection and intravesical instillation, were compared. The concentration of CASPc in bladder and bladder tumor after intravenous injection and intravesical instillation was similar. The ratio of dye uptake between tumor and normal bladder after either administration was approximately two. Although no systemic absorption of the photosensitizer was observed after intravesical instillation, there was no reduction in tumor uptake or in the ratio between tumor to normal surrounding tissue. Therefore, no systemic side effects or skin phototoxicity are expected upon intravesical instillation.The microscopic biodistribution of CASPc after intravenous injection and intravesical instillation was also compared. After intravenous injection, the photosensitizer was distributed within the whole tumor with increased fluorescence around the microvasculature. In the normal bladder wall, weak fluorescence was seen in the area of the vasculature in the submucosa and the muscularis. After intravesical instillation, strong fluorescence was detected only at the tumor surface and in normal urothelium; no fluorescence was found in other areas of the tumor or in submucosa or muscularis.A comparison of the photodynamic treatment of model bladder tumors showed that tumor destruction after either method was similar but that there were less side effects to normal bladder wall after intravesical instillation of the CASPc. Intravesical administration of photosensitizers may, therefore, be a viable alternative to intravenous injection with potential for reduced systemic and normal tissue toxicity.

Photodynamic therapy of experimental subchoroidal melanoma using chloroaluminum sulfonated phthalocyanine.

Although photodynamic therapy has shown great promise for the treatment of a variety of malignant neoplasms, the role of this new therapeutic modality in the clinical management of intraocular tumors remains incompletely understood. This study examines the effects of photodynamic therapy using chloroaluminum sulfonated phthalocyanine on Greene hamster melanoma transplanted into the subchoroidal space in rabbits. Twenty-four hours after intravenous administration of chloroaluminum sulfonated phthalocyanine (5 mg/kg), tumors were irradiated with 675 nm of light at total light doses of 7 to 60 J/cm2. The results show that tumor growth was arrested at total light doses of 22 to 60 J/cm2. At total light doses of 15 to 21 J/cm2, tumor growth was initially arrested. However, regrowth of these tumors was apparent within 7 days. Total light doses of less than 15 J/cm2 showed no response. Complications of photodynamic therapy, such as intraretinal or subretinal hemorrhages and retinal detachment, were seen only in animals who received total light doses in excess of 43 J/cm2.

Photodynamic therapy of arteries. A novel approach for treatment of experimental intimal hyperplasia.

Photodynamic therapy (PDT) uses light activation of otherwise nontoxic dyes for the production of reactive oxygen species that cause cell injury and death. The inhibition of intimal hyperplasia (IH) by PDT was studied in the balloon injury model of the rat carotid artery. Chloroaluminum-sulfonated phthalocyanine (CASPc) was the drug chosen for PDT because it does not produce skin photosensitivity and has a high absorption peak of light at 675 nm, a wavelength with good tissue penetration. A pilot study indicated that CASPc administration with laser radiant exposure of 100 J/cm2 resulted in a homogeneous, circumferential effect on the whole artery. Male Sprague-Dawley rats received the balloon catheter injury to the left common carotid artery (day 0) and were equally divided into two groups. Nine rats received either CASPc (5 mg/kg i.v., n = 6) or saline (n = 3) at day 2, before IH was present, and nine rats received CASPc or saline in the same manner on day 7, when IH was already present. Twenty minutes after drug injection, the distal left common carotid artery was irradiated under saline with 675-nm laser light at 100 mW/cm2 for 10(3) seconds (100 J/cm2). At this low laser irradiance, there are no thermal effects, but photoactivation of CASPc occurs. The rats were killed at day 14 after balloon injury when IH reaches a maximum. The arteries were harvested after perfusion-fixation for light microscopy, histological and computerized morphometric evaluation, and transmission electron microscopy (TEM) analysis. The cross-sectional areas of the neointima were measured in the PDT-treated arteries and in the laser-only control arteries. There was a significant mean +/- SD decrease of IH in the PDT-irradiated segments of the arteries (0.06 +/- 0.05 mm2) versus the laser-only control ones (0.17 +/- 0.07 mm2) (t test, p less than 0.001), with no statistical difference between the day 2 and day 7 treated rats. Lack of IH was correlated in 90% of cases with histological absence of medial smooth muscle cells or inflammatory cells, but no other structural injury was identified. TEM analysis showed early evidence of PDT-mediated cytotoxic effects at 4 hours and the absence of collagen or elastic tissue structural alterations. These data demonstrated that PDT can effectively inhibit the IH response when it is used before or during induction of cellular proliferation in this acute model. Although the long-term implications of PDT in arteries need to be defined, this technique may offer a new method for understanding and treating IH.

Effect of aspirin on photodynamic therapy utilizing chloroaluminum sulfonated phthalocyanine (CASP).

The efficacy of photodynamic therapy (PDT) is mediated through a direct vascular effect. Interference with platelet function and resulting vascular stasis have been recently demonstrated utilizing the photosensitizer dihematoporphyrin ether (DHE). We examined the effect of aspirin, a known inhibitor of both cyclooxygenase and platelet activity, on PDT using chloroaluminum sulfonated phthalocyanine (CASP). Thirty-six rats implanted with a window chamber were given either 0.1 mg/kg (low dose) or 10 mg/kg (high dose) aspirin immediately before, immediately after, or 6 hours after the completion of CASP-PDT. Aspirin in either dosage did not appear to have any effect on the window vasculature when given immediately after light exposure. A moderate inhibition of vascular response was seen in animals treated with aspirin pre-PDT, whereas high-dose aspirin completely abrogated the CASP-PDT vascular response when given 6 hours post-PDT. These data indicate that aspirin can effect CASP-PDT in both time-dependent and dose-dependent fashions.

Chloroaluminum sulfonated phthalocyanine versus dihematoporphyrin ether: Early vascular events in the rat window chamber

The development of a simple and well-tolerated rat window chamber has allowed direct comparison of the vascular effects of two photosensitizers, chloroaluminum sulfonated phthalocyanine (CASP) and dihematoporphyrin ether (DHE). CASP and DHE were given 4 days after the implantation of the window chamber. Photodynamic therapy (PDT) with CASP was performed 24 hours after intravenous injection (10 mg/kg) with light at 675 nm (power density 200 mW/cm2, incident energy 100 J). DHE was given in a similar fashion (5 mg/kg intraperitoneally; light at 630 nm with matching power density and energy settings 24 hours after injection). Using videomicroscopic and integrating sphere measurements, marked differences were noted in the vascular effects of these photosensitizers. DHE caused immediate hemorrhage and disruption of the postcapillary venules, while CASP induced vascular spasm starting 4 hours after the completion of PDT. Forty-eight hours after PDT, both systems demonstrated a loss of chamber-induced neovascularization.