Christoph Abels

Effects of photodynamic therapy on leucocyte-endothelium interaction : differences between normal and tumour tissue

An inflammatory reaction is regularly noticed in irradiated tissues following photodynamic therapy (PDT). This observation is potentially associated with leucocyte-mediated tissue damage, which might further contribute to the tumoricidal effect of this therapy. The objective of our study was to investigate the effects of PDT on leucocyte-endothelium interaction in the microvasculature of tumours and normal tissue. Experiments were performed in the dorsal skinfold chamber preparation of Syrian golden hamsters bearing amelanotic melanoma A-Mel-3. The photosensitiser. Photofrin (5 mg kg-1 i.v.) was injected 24 h before laser irradiation (630 nm, 100 mW cm-2, 10 J cm-2 or 100 J cm-2). Post-capillary confluent venules (diameter 15-40 microns) of subcutaneous (s.c.) tissue or the amelanotic melanoma A-Mel-3 were observed by intravital microscopy before, 5, 30, 60 and 180 min after laser irradiation and recorded for off-line analysis. Before treatment, the number of adherent leucocytes in tumour vessels was only 22% of the number observed in vessels of s.c. tissue (P < 0.01). The maximum increase in adhering leucocytes was observed in post-capillary venules of s.c. tissue 1 h after PDT (P < 0.01). In contrast, enhanced leucocyte-endothelium interaction was missing in tumour vessels and in control groups. These results indicate that the tumour destruction observed after PDT is not mediated by leucocyte-endothelium interaction in the tumour. Induction of leucocyte adhesion in the PDT-treated normal tissue suggests a contribution to the peritumoral inflammatory response. Different maturational status or biochemical properties of tumour microvascular endothelium may explain the lack of leucocyte adherence upon PDT.

Pharmacokinetics and selectivity of aminolevulinic acid–induced porphyrin synthesis in patients with cervical intra-epithelial neoplasia

Photodynamic therapy (PDT), due to its tumor selectivity, represents an alternative approach to diagnose and treat cervical intra‐epithelial neoplasia (CIN) without altering normal surrounding tissue. Our aim was to investigate the pharmacokinetics and the selectivity of 5‐aminolevulinic acid (5‐ALA)–induced porphyrin fluorescence after topical administration, to obtain basic clinical data for future diagnostic fluorescence imaging and PDT protocols for CIN. Twenty‐eight non‐pregnant women with a cytological diagnosis of low‐grade or high‐grade squamous intra‐epithelial lesions were included. An aqueous solution containing 3% 5‐ALA was topically applied 1 to 6 hrs prior to conization using a cervical cap. After excision, porphyrin‐induced fluorescence was quantified in dysplastic (n = 14) and normal epithelium (n = 28) by means of quantitative fluorescence microscopy. High values of porphyrin fluorescence were found in squamous epithelium between 150 and 450 min, with a maximum at 300 min following administration of 5‐ALA. Ratios of porphyrin fluorescence of dysplastic vs. surrounding normal epithelium were 1.3 and 1.21 for CIN 1 (n = 3) and CIN 2 (n = 3), respectively. In CIN 3 patients (n = 8), this ratio was 2.35; the best selectivity of 5‐ALA‐induced porphyrin fluorescence in CIN 3 lesions (ratio 3) was observed with a topical administration time of between 150 and 250 min. Our results demonstrate that patients with CIN 3 show higher 5‐ALA‐induced fluorescence compared with normal epithelium. The optimal administration time of topically applied 5‐ALA was between 3 and 4 hr. Our data suggest that topical ALA‐PDT and photodynamic diagnosis might be suitable for detecting CIN. Int. J. Cancer 78:310–314, 1998.© 1998 Wiley‐Liss, Inc.

Photodynamic therapy with 5-aminolaevulinic acid-induced porphyrins of an amelanotic melanoma in vivo

Of particular interest for photodynamic therapy (PDT) are the endogenously formed and photodynamically active porphyrins produced following topical or systemic application of 65-aminolaevulinic acid (ALA), a haem precursor. Having determined the pharmacokinetics and wavelength dependence of PDT with ALA-induced porphyrins, we analysed the porphyrin metabolites in tumour and surrounding skin. The therapeutic efficacy of PDT using ALA-induced porphyrins was investigated. Amelanotic melanomas (A-Mel-3) were implanted subcutaneously in the back of Syrian golden hamsters (body weight (b.w.), 70-80 g). After 5-7 days, tumours with a volume of approximately 150 mm3 were used for PDT (n = 36). ALA (500 mg kg-1 b.w., pH 6.5) was injected intravenously 45, 90, 150 and 300 min before light irradiation (635 nm, 100 mW cm-2, 100 J cm-2). Tumours with light irradiation only served as controls. The tumour volume was measured after PDT for 28 days. The total porphyrin content was determined in the tumours, the surrounding skin and erythrocytes prior to and 45, 90, 180, 240, 300 and 480 min and 24 h following intravenous injection of ALA (500 mg kg-1 b.w.; n = 32). Porphyrin metabolites were separated by high pressure liquid chromatography (HPLC). Tumour growth was significantly delayed when PDT with ALA was performed 45, 90 or 150 min following intravenous administration. At that time, protoporphyrin (1.8 +/- 0.4 nmol g-1), coproporphyrin (2.2 +/- 0.5 nmol g-1) and uroporphyrin (1.7 +/- 1.4 nmol g-1) were the main metabolites in the tumour tissue. Erythrocytes also contained significant amounts of porphyrins (11.8 +/- 1.3 nmol g-1). The tumour and surrounding skin exhibited a different pattern of porphyrin metabolites. Unexpectedly, a single treatment of PDT with ALA-induced porphyrin resulted in only one complete remission out of six amelanotic melanomas when the final therapeutic outcome was assessed after 28 days. The therapeutic efficacy of PDT with ALA-induced porphyrins can be positively correlated with the fluorescence kinetics previously determined. The analysis of the porphyrin metabolites in amelanotic melanoma by HPLC indicates that the porphyrin accumulation is not due to a decreased activity of ferrochelatase. Moreover, the photodynamic effects may not be mediated solely by porphyrins localized in the tumour parenchyma, but also by significant amounts of porphyrins in the microvasculature. PDT with this endogenous photosensitizer failed to induce complete emission of the treated tumours despite irradiation at the time of maximum porphyrin concentration using the optimum therapeutic wavelength. Thus PDT with ALA-induced porphyrins is less effective in our model relative to that observed for the exogenous photosensitizer Photofrin or synthetic porphycenes after a single treatment.

Characterization and prevention of phototoxic effects in intravital fluorescence microscopy in the hamster dorsal skinfold model

Intravital microscopy is widely used to study the microcirculation. However, the use of fluorescent dyes can induce phototoxic effects which may affect the measurements, particularly in tissue exposed to oxidative stress. The aim of the study was to determine the threshold light dose at which fluorescent microscopy is associated with phototoxic effects in the hamster dorsal skinfold chamber under normal and pathological conditions. The extent of phototoxicity in the microcirculation in the hamster skinfold chamber was investigated using intravital fluorescent microscopy during 60 min of illumination (1048 mW/cm2) applying two different concentrations of fluorescein isothiocyanate dextran under baseline conditions (groups A and B) and following 4 h of ischemia (groups C and D). In the second part of the study the microvasculature was analyzed regarding phototoxic effects during a standardized intravital microscopic examination after 4 h of pressure induced ischemia. Groups I and II (n=7) were studied using epiillumination after injection of fluorescein isothiocyanate dextran plus rhodamine 6G or rhodamine 6G only. In group III (n=7) only transillumination was used. Arteriolar vasospasm, microvascular perfusion failure, thrombus formation, and enhanced leukocyte endothelium interaction were observed as signs of a phototoxic effect in normal tissue. However, the light doses needed to induce these effects clearly exceeded those during standard examinations. The induction of a 4-h ischemia and reperfusion further enhanced these effects. Despite the predamage by ischemia/reperfusion the comparison of epiillumination and transillumination microscopy using a standard protocol showed no differences regarding the parameters analyzed at any time. This indicates that epiillumination and the fluorescent dyes per se did not affect the experimental results. These results show that ischemia/reperfusion studies in the dorsal skinfold chamber of the Syrian golden hamster can be carried out safely without the risk of inducing phototoxic effects by fluorescent microscopy. Nevertheless every laboratory using epiillumination and fluorescent dyes should take precautions to avoid these effects by the use of sensitive cameras to lower the light dose

Targeting of the tumor microcirculation by photodynamic therapy with a synthetic porphycene

9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) is a chemically pure substance with fast pharmacokinetics and superior photodynamic properties in vitro as compared to Photofrin. In this study the pharmacokinetics, photodynamic efficacy and tissue localization of ATMPn were investigated in vivo. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters. Fluorescence kinetics of ATMPn (1.4 mumol kg-1 b.w.i.v.; n = 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 mumol kg-1 was injected 24 h (n = 3), 3 h (n = 3), 1 min (n = 6) and 2.8 mumol kg-1 1 min (n = 6) before PDT (Laser (630 nm) or lamp (600-750 nm), 100 mW cm-2, 100 J cm-2). Tumor volume was measured for 28 d. Solid tumors (n = 3) were excised 1 min after injection of ATMPn (2.8 mumol kg-1) and cryostat sections (20 mm) were analyzed by confocal laser scanning microscopy (CLSM) for tissue localization of the dye. Maximal fluorescence (mean +/- S.E.) arose in the tumor (94 +/- 7%) and surrounding host tissue (67 +/- 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable 12 h after administration. Only PDT 1 min after injection of ATMPn was effective yielding 3/6 complete remissions (2.8 mmol kg-1, laser) and 6/6 complete remissions (2.8 mumol kg-1, lamp), respectively. One minute after injection the dye is primarily localized in the vascular wall of normal and tumor vessels as shown by CLSM. PDT at a time, when the dye is localized primarily in the tumor microcirculation, exhibits the best tumor killing effects showing that vascular targeting is effective in treating solid malignant tumors. ATMPn in liposomes makes administration and light irradiation in one session possible due to its fast pharmacokinetics. Thus, using ATMPn as a photosensitizer may provide more flexibility to perform PDT after surgical exploration and debulking as adjuvant therapy.

IMPACT OF DEXTRAN ON MICROVASCULAR DISTURBANCES AND TISSUE INJURY FOLLOWING ISCHEMIA/REPERFUSION IN STRIATED MUSCLE

ABSTRACT The aim of this study was to evaluate the effect of dextran (Dx) 1 versus Dx 60 (molecular weights 1,000 and 60,000) on microvascular disturbances and tissue injury in striated muscle after ischemia/reperfusion (I/R). Experiments were performed using a 4 h pressure-induced ischemia model in the hamster dorsal skinfold chamber. Three groups (n = 6) of animals received a continuous infusion (45 min, 3 ±L/mon) of either Dx 1 or Dx 60 (total dose 5 mg/kg) or saline solution beginning 15 min before reperfusion. Intravital fluorescence microscopy allowed for quantification of functional capillary density, leukocyte adherence, extravasation of fluorescein isothiocyanate-Dx, and nonviable (propidium-positive) cell count before ischemia and .5, 2, and 24 h after reperfusion. Experiments were terminated with tissue preservation for electron microscopy. Postischemic functional capillary density was significantly improved by Dx 60 (at 24 h, 88% vs. 51 % in controls). In animals receiving postischemic Dx 1 or Dx 60, leukocyte adherence was significantly reduced (at .5 h, 44% and 58%, respectively) as compared with controls, whereas macromolecular extravasation was unchanged. Nonviable cell count was significantly decreased by both Dx fractions (at 24 h, Dx 1, 75%; Dx 60, 87%), indicating a reduction of tissue injury, which was also confirmed by electron microscopy. These results provide evidence that Dx 60 at 5 mg/kg attenuates I/R injury more effectively than Dx 1. Leukocytes play a major role in the development of I/R injury, but macromolecular extravasation does not always correlate with the leukocyte-endothelium interaction and the manifestation of I/R injury.

Chapter 1 History of photodynamic therapy in dermatology

Abstract A “photodynamic reaction” describes a photochemical process involving the absorption of light by a photosensitizer and the subsequent generation of reactive oxygen species. Hermann von Tappeiner coined the term “photodynamic” after numerous experiments in 1904 in order to distinguish this photooxidative process from the sensitization during photography. Already at this early stage patients with dermatological conditions like lupus vulgaris or basal cell carcinoma were treated by von Tappeiner in cooperation with the dermatologist A. Jesionek with PDT. However, it took over 90 years until photosensitizers were approved, first in disciplines like pulmonology or gastroenterology. Although dermatology was the discipline, were the very first patients were treated, PDT was not approved until 1999, when in the US 5-aminolevulinic acid for PDT of actinic keratoses was registered.

Photodynamische Therapie epithelialer Präkanzerosen und Karzinome

Bereits vor 100 Jahren, im Wintersemester 1897/98, wurde der photodynamische Effekt durch den Munchner Medizinstudenten Oscar Raab im Rahmen seiner Dissertation entdeckt [57] und im folgenden zur Behandlung von Hauttumoren eingesetzt [34, 71]. Die Wirksamkeit der photodynamischen Therapie (PDT) in der Behandlung oberflachlicher Prakanzerosen und Kanzerosen der Haut wurde zwischenzeitlich ausfuhrlich beschrieben und dokumentiert [15, 17, 20, 39, 49, 69, 79]. Die Zulassung der PDT erfolgte allerdings erst fur die Indikationen Blase-, Osophagus- und Lungentumoren in Japan, Kanada, USA und den Niederlanden.

Targeting of the tumor microcirculation with a new photosensitizer

Tumors are characterized by an insufficient neoangiogenesis. Therefore targeting of the fragile tumor microcirculation by photodynamic therapy (PDT) may induce easily tumor ischemia leading to tumor necrosis. Nine-acetoxy-2,7,12,17-tetrakis-((beta) -methoxyethyl)- prophycene (ATMPn) is a chemically pure, lipophilic substance and revealed superior photodynamic characteristics in vitro as compared to PhotofrinR. In this study pharmacokinetics, photodynamic effects and localization of ATMPn incorporated in small unilamellar liposomes in tumor and surrounding normal tissue were evaluated. Amelanotic melanomas (A-Mel-3) were implanted in dorsal skin fold chambers fitted to Syrian Golden hamsters (70 - 80 g b.w.). Fluorescence kinetics of ATMPn administered intravenously (1.4 micrometers ol/kg b.w.; n equals 8) were monitored by intravital microscopy. Quantitative measurements of fluorescence intensity were carried out by digital image analysis. For tumor growth studies 1.4 micrometers ol/kg was injected 24 h (n equals 3), 3 h (n equals 3), 1 min (n equals 6) and 2.8 micrometers ol/kg 1 min (n equals 6) before PDT (630 nm, 100 mW/cm2, 100 J/cm2). Tumor growth was measured over 28 days. Solid tumors (n equals 3) were excised 1 min after injection of ATMPn (1.4 micrometers ol/kg) and cryostat sections (10 micrometers) were analyzed by confocal laser scanning microscopy (CSLM) to determine tissue localization of dye. Maximal fluorescence (mean plus or minus S.E.) arose in tumor (94 plus or minus 7%) and surrounding host tissue (67 plus or minus 5%) 30 s post injection followed by a rapid decrease. Hardly any fluorescence was detectable after 12 h. Only PDT 1 min after injection of ATMPn was effective yielding 1/6 complete remission (1.4 micrometers ol/kg) and 3/6 complete remissions (2.8 mmol/kg), respectively. At that time dye is primarily localized in vessels and vessel walls as shown by CSLM. ATMPn in liposomes reveals very rapid kinetics thus suitable for intraoperative PDT. Moreover, PDT (2.8 micrometers ol/kg) at time, when dye is localized in tumor microcirculation, exhibits best tumor killing effects.

Wavelength-dependent in-vitro and in-vivo photodynamic effects after sensitization with 5-aminolevulinic acid induced protoporphyrin IX

Photodynamic therapy (PDT) with topically applied 5-aminolevulinic acid (ALA) is of growing interest, in particular in dermatology. Due to the fact that PDT with intravenously administered Photofrin is the only clinically approved sensitizer so far and is performed at a wavelength of 630 nm, this wavelength is also used in most experimental and clinical trials with ALA. In this study influence of irradiation with coherent light from a tunable dye laser at different wavelengths ranging from 625 to 649 nm was investigated. In in vitro experiments HaCaT immortalized human keratinocytes were sensitized with 30 (mu) g/ml ALA for 24 hrs. By determination of cell viability with the MTT test, best cell-killing effects were observed following irradiation at 635 nm. In an in vivo setting using an amelanotic melanoma (A-Mel-3) grown subcutaneously in Syrian Golden hamsters, these results were confirmed: tumor growth determined by measuring tumor volume increase after 28 days was less pronounced in animals treated with 100 mg/kg ALA i.v. and irradiated 2.5 hrs. later at 635 nm, as compared to animals receiving an equal dose and irradiated at 630 nm. This observation in vitro is probably due to large amounts of photosensitizing protoporphyrin IX (PP) localized in cell membranes which is visualized by confocal laser scanning microscopy (CLSM) and determined by HPLC analysis. These results suggest that in ALA-PDT when a coherent light source is used probably better results are achieved irradiating at 635 nm.