Robert D. Bower

WAVELENGTH-DEPENDENT EFFECTS OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A in vivo AND in vitro

Abstract Benzoporphyrin derivative monoacid ring A (BPD‐MA) is a chlorin‐like photosensitizer currently in clinical trials for cancer and psoriasis. It has maximal absorption peaks at both 630 and 690 nm and can be activated at both these wavelengths. In vitro phototoxicity tests using the P8 15 murine mastocytoma cell lines conducted over wavelengths of light between 678 and 700 nm emitted by an argon‐ion pumped dye laser showed that equivalent cell kill could be achieved between 682 and 690 nm. Tests on in vivo phototoxicity of normal skin of DBN2 mice injected with 2 mg/kg of BPD‐MA and exposed to light at 125 J/cm2, between 620 and 700 nm, demonstrated peaks of normal skin damage occurring at 630–640 nm and 680–690 nm. In tests carried out with light between 620 and 700 nm, at 10 nm increments, it was seen that light delivered at 680–690 nm caused slightly more damage to normal skin than light delivered at 630–640 nm. When lower doses of light between 675 and 705 nm were tested using smaller increments, it was determined that equivalent skin damage occurred over a range of 68–95 nm. Antitumor efficacy in tumor‐bearing DBN2 mice was tested between 683 and 695 nm. It was found that equivalent antitumor efficacy, determined by assessing tumor‐free status at 20 days posttreatment, occurred at wavelengths between 685 and 693 nm. When tumor‐bearing animals injected with BPD‐MA at 2 mdkg and exposed to light 3 h later were treated with either 630 or 690 nm light at various doses, it was observed that 690 nm light was more effective at tumor ablation than was 630 nm light, demonstrating that while similar damage to normal skin may be effected by equivalent doses of light at either wavelength, tumor ablation was greater at 690 nm. Further, our data suggest that alternative light sources with bandwidths greater than those of the argon‐ion pumped dye laser (±0.3 nm) may have equivalent efficacy with this photosensitizer.

Preventing Intimal Hyperplasia With Photodynamic Therapy Using an Intravascular Probe

The purpose of this study was to determine the efficacy of intravascular photodynamic therapy (PDT) to prevent the development of intimal hyperplasia. Anesthetized New Zealand white rabbits underwent placement of Fogarty balloon catheters introduced via femoral artery cutdowns. Catheters were passed retrograde 10 cm into the lower abdominal aorta, inflated six times, and withdrawn toward the inguinal ligament. Rabbits were then randomly assigned to one of the following groups: group 1, drug with no light; group 2, no drug with 240 joules of light; group 3, drug plus 120 joules of light; or group 4, drug plus 240 joules of light. Uninjured carotid arteries served as negative control vessels (N) and injured but non-PDT-treated iliac artery segments served as positive controls (P). Porfimer sodium (Photofrin) was administered in a dose of 5.0 mg/kg. Light was provided by a fiberoptic probe with a 1 cm cylindric diffuser attached to an argon pumped dye laser tuned to 630 nm to provide 1 W of laser light for 120 or 240 seconds. One month after PDT, rabbits were killed, perfusion fixed with glutaraldehyde, and vessels removed and examined microscopically. Intimal thickness (mean ± SD) was calculated and expressed as ratios of the intima/media at four equal positions. Results for N, P, and groups 1, 2, 3, and 4 were 0.02±0.00, 1.18±0.71, 0.76±0.33, 0.96±0.43, 0.14±0.22, and 0.36±0.16, respectively. Intimal thickness was significantly reduced in groups 3 and 4 when compared with P, group 1, and group 2 (p<0.001, ANOVA). These results showed that intravascular PDT was effective in reducing intimal hyperplasia following arterial injury. This may be a practical method of delivering light for PDT.

Determining light dose for photodynamic therapy of atherosclerotic lesions in the Yucatan miniswine.

Purpose: To determine the light dose required for photodynamic therapy of atherosclerotic lesions in the miniswine. Methods: Aortic atherosclerosis was created in seven Yucatan miniswine by a combination of balloon endothelial injury and 2% cholesterol and 15% lard for 7 weeks. Six animals received the photosensitizer PhotofrinR 2.5 mg/kg, while an additional swine received no drug. After 24 hours, the abdominal aorta was exposed and the aorta opened longitudinally in each animal. Three 1-cm spots were illuminated with energy densities of 60, 120, and 240 J/cm2 from an argon-pumped dye laser tuned to 630 nm with a laser output of 1 W. Four weeks later, the animals were killed, abdominal aortae removed, and intimal thickness determined by morphometry. Results: The percentage intimal thickness (mean ± SD) was 36.7 ± 27.1, 9.1 ± 5.0, and 6.4 ± 8.1 for the three energy densities, respectively. Although both 120 and 240 J/cm2 energy densities produced significant (p < 0.05) reduction in atheroma, considerable damage to the underlying media was also observed in the 240 J/cm2 group. Conclusions: A Photofrin dose of 2.5 mg/kg and 120 J/cm2 light are necessary for adequate ablation of atheroma while avoiding extensive medial damage.

Preventing restenosis in atherosclerotic miniswine with photodynamic therapy

The purpose of this study was to determine whether the addition of Photodynamic Therapy (PDT) using the photosensitizer Photofrin* (P*) following balloon angioplasty (BA) could prevent restenosis in an atherosclerotic animal model. Bilateral iliac atherosclerosis was created in 21 Yucatan miniswine. Six weeks later, P* 2.5 mg/kg was given IV 24 hours prior to BA (4 mm X 20 mm, 1 inflation). Following BA, swine were randomly allocated to receive PDT via a fiberoptic probe with laser energy or the same probe without laser energy. The fiberoptic probe had a 1 cm cylindrical diffusing tip and was passed co-axially through a custom catheter to ensure central location of the probe. A continuous wave argon ion-pumped dye laser tuned to 630 nm was used to provide a fluence of 100 J/cm2. Four weeks later, swine were sacrificed and vessels perfusion-fixed in-situ with glutaraldehyde and analyzed by ocular micrometry. Five occlusions occurred, all in the PDT + BA group. Percentage intimal thickness (mean +/- SD) was 51.0 +/- 29.5 in the BA group and 71.2 +/- 35.2 in the BA + PDT group (p equals 0.21). These results suggest that the addition of PDT following BA does not prevent restenosis.

Photodynamic treatment with BPD-MA (verteporfin) activated with light within different spectral ranges

Benzoporphyrin derivative monoacid ring A [BPD-MA (verteporfin) or BPD], a second generation photosensitizer tested in clinical trials in combination with red light was compared for its PDT efficiency in vitro and in vivo upon activation with light in the UVA, blue and red spectral ranges. PDT efficiency, calculated based on the BPD absorption spectrum and spectral output of the different light sources, was compared with actual PDT efficiency determined in vitro and in vivo. Results obtained in an in vitro cytotoxicity assay, in which aliquots of murine P815 cells, pre-incubated for 1 h with BPD at 5 ng/mL, were exposed simultaneously to various light doses delivered within UVA, blue and red spectral ranges showed that in this test system PDT efficiency was governed by BPD absorption and light source emission spectra. Similar results were obtained in an in vitro BPD photobleaching test. Thus in vitro, values for calculated, theoretical PDT efficiency corresponded to the actual PDT efficiency. However, in vivo factors, such as depth of tissue penetration with light and localization of the target, had an important influence on PDT efficiency. In mouse models of skin photosensitivity and the cutaneous hypersensitivity immune response (CHS) assay, because of the thinness of mouse skin, PDT efficiency approximated the theoretical PDT efficiency, although blue light was somewhat more efficient in PDT than UVA, and red light was somewhat more efficient than blue or UVA. In a pig skin photosensitivity model, red light induced the highest skin response manifested by erythema and swelling, while blue light caused erythema and minimal swelling and UVA caused only erythema. These differences could be related to the thickness of pig skin and the depth of tissue penetration characteristic of each spectral range. Fluence rate was found to be an additional factor which modifies the effect of BPD and light. In conclusion, BPD can be efficiently activated with light within the UVA, blue and red spectral ranges. Moreover, light doses, deemed safe for red light, can be utilized with light of other spectral ranges, but only after a very careful evaluation of the conditions under which they were determined and the conditions under which they will be used.

Photodynamic therapy using Verteporfin and 630-nm laser light in canine esophagus

Objective: The goal ofthis study was to determine a relationship between light dose and the delay time between administration of Verteporfin and light to ablate canine esophageal mucosa. Materials and Methods: Verteporfin was administered IV (0.75 mg/lg). 630-nm light from KTP/Dye laser was delivered using a 9-cm diffuser inside a specially designed reflective 7-cm windowed balloon. Initially, animals were treated at doses of 160 J/cm, 180 J/cm, 200 J/cm, and 220 J/cm 2-3 hours after Verteporfin injection. Based on the acute response in these animals and the plasma Verteporfin clearance, other treatments were devised and tested at 60 J/cm (at 1 5 minutes), 80 J/cm (22 minutes), 100 J/cm (30 minutes), and 145 J/cm (60 minutes). Results: In initial animals, 200 J/cm at 2 hours induced acceptable mucosal ablation. Using this light dose and the plasma drug clearance, subsequent treatments were tested resulting in similar injuries using 60 3/cm (15 minutes), 80 3/cm (22 minutes), 100 3/cm (30 minutes), and 145 J/cm (60 minutes). Conclusions: A relationship was established relating the light dose and the delay time between administration oflight and Verteporfin for ablation ofnormal canine esophageal mucosa.

Selective treatment of atherosclerosis using photodynamic therapy in the Yucatan miniswine: preliminary results

Photodynamic therapy (PDT) represents a novel method of selectively treating atherosclerosis using a combination of photosensitizer drug, low power laser light, and molecular oxygen. In this preliminary study, PDT was used to treat atherosclerotic lesions in a miniswine model. Yucatan miniswine weighing between 20-30 kg, were rendered atherosclerotic by a combination of balloon endothelial injury and dietary supplementation with 2% cholesterol and 15% lard diet for 7 weeks. Following this, miniswine were given a porphyrin-type photosensitizer, Photofrin 2.5 mg/kg IV. Twenty-four hours after receiving Photofrin, swine received a general anesthetic and the infrarenal abdominal aorta was exposed. Through a longitudinal aortotomy, the posterior aortic wall was irradiated with 630 nm laser light at one of the following light doses: 60, 120, and 240 J/cm2. Four weeks after PDT, swine were killed and perfusion- fixed with glutaraldehyde. Light microscopy showed a decrease in intimal thickness for all light doses. Decreased cellular elements were seen in the irradiated zones as the laser power was increased. Non-irradiated sites showed typical atherosclerotic lesions with foam cells, fibrosis and calcification. This study demonstrated the feasibility of using PDT for atherosclerotic lesions.