Protoporphyrin IX fluorescence kinetics in UV-induced tumours and normal skin of hairless mice after topical application of 5-aminolevulinic acid methyl ester

Accumulation of protoporphyrin IX (PpIX) was investigated in normal skin and UV-induced tumours in hairless mice after topical application of a cream containing 2, 8 or 16% of 5-aminolevulinic acid methyl ester (ALA-Me). Higher levels of PpIX were measured in tumours compared to normal skin. The maximal amount of PpIX was reached at 1.5, 3 and 4 h after 2, 8 and 16% ALA-Me application, respectively. Higher tumour to normal skin PpIX fluorescence ratios were measured after application of 8 and 16% ALA-Me than after application of 2%. After irradiation with a broad spectrum of visible light from a slide projector, more than 90% of PpIX was bleached by fluences of 36 and 48 J/cm2, at fluence rates of 10 and 40 mW/cm2 respectively. At these fluences, the PpIX photobleaching rate was significantly higher (P<0.05) in normal mouse skin than in tumours. In addition, for a given fluence, more PpIX was photobleached at the lower fluence rate (10 mW/cm2) than at the higher fluence rate (40 mW/cm2) in normal skin (P<0.001) as well as in tumours (P<0.05) after exposure to 24 J/cm2 of light. In conclusion, the highest tumour to normal skin PpIX ratio was observed 3 h after application of 8% ALA-Me, suggesting that light exposure should be performed at this time in order to achieve an optimal PDT effect in this tumour model.     

Photodynamic Therapy of 9L Gliosarcoma with Liposome-Delivered Photofrin

Abstract— The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm₂ of 632 nm light at 100 mW/cm₂) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor ( P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher ( P = 0.021) using liposome (33.8 ± 18.9 μg/g) compared to dextrose delivery (5.5 ± 1.5 μg/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.     

Subcellular Localization of Photofrin and Aminolevulinic Acid and Photodynamic Cross-Resistance in Vitro in Radiation-Induced Fibrosarcoma Cells Sensitive or Resistant to Photofrin-Mediated Photodynamic Therapy

Abstract— The subcellular and, specifically, mitochondrial localization of the photodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induced protoporphyrin-IX (PpIX) has been investigated in vitro in radiation-induced fibrosarcoma (RIF) tumor cells. Comparisons were made of parental RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-mediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochondria-specific probes 10N-Nonyl acridine orange (NAO) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization of Photofrin and PpIX with Rh-123 was determined by double-label confocal fluorescence microscopy. Clonogenic cell survival after ALA-mediated PDT was determined in RIF-1 and RIF-8A cells to investigate cross-resistance with Photofrin-mediated PDT. At long (18 h) Photofrin incubation times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-1 than in RIF-8A cells. Differences between RIF-1 and RIF-8A in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofrin binding site. The differences in this binding may account for the PDT resistance in RIF-8A cells. With ALA, the peak accumulations of PpIX occurred at 5 h for both cells, and followed a diffuse cytoplasmic distribution compared to mitochondrial localization at 1 h ALA incubation. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localization in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cross-resistance to incubation in PpIX but not to ALA-induced PpIX, implying differences in mitochondrial localization and/or binding, depending on the source of the PpIX within the cells.     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.     

Biodistribution of Photofrin II® and 5-Aminolevulinic Acid-Induced Protoporphyrin IX in Normal Rat Bladder and Bladder Tumor Models: Implications for Photodynamic Therapy

Photodynamic therapy (PDT) has been considered as a potential therapy for superficial bladder carcinomas. Cutaneous photosensitivity and reduction of bladder capacity are the two well-known complications following systemic administration of the commonly used photosensitizer, Photofrin II® (PII). The objective of the present study was to evaluate whether intravesical. (i.b.) instillation of photosensitizers for PDT of bladder cancer might be a more suitable treatment method. Female Fischer rats were utilized to develop orthotopic and heterotopic bladder tumor models. Rats bearing orthotopic bladder tumors were treated either intravesically or intravenously with graded doses of 5-aminolevulinic acid (ALA) or PII. Normal rats received the same doses of ALA or PII. As well, rats bearing heterotopic tumor were studied for comparison. The biodistribution times (times allowed for tissue uptake and bioconversion following drug administration) were 2, 4 or 6 h. Porphyrin fluorescence intensities within tumor, urothelium, submucosa, bladder muscularis and abdominal muscle were quantitated by confocal laser scanning microscopy. Following intravenous (i.v.) injection of ALA, tumor protoporphyrin IX (PpIX) levels peaked at 4 h and diminished by 6 h. The PpIX ratios of tumor-to-bladder mucosa, submucosa and muscle layers were 3:1, 5:1 and 8:1, respectively, 4 h following 1000 mg/kg ALA injection. After ALA instillation, the optimal biodistribution time appeared to be 4 h. Bladder instillation provided comparable tumor labeling with the i.v. route, but lost selectivity of PpIX accumulation between tumor and normal urothelium. The PpIX ratio of tumor-to-bladder muscularis was 5:1. After i.b. instillation of PII, porphyrin fluorescence was detected only within tumor and urothelium, while porphyrin fluorescence was mainly located in bladder submucosa following i.v. injection. Intravesical administration of ALA or PII might be feasible for PDT of superficial bladder cancers.     

Photo‐co‐carcinogenesis of Topically Applied Retinyl Palmitate in SKH‐1 Hairless Mice

Cosmetic products that contain retinyl palmitate are popular as antiaging skin treatments; however, recent studies suggest a risk for enhanced skin tumor development with topical retinyl palmitate applications and exposure to solar ultraviolet radiation (UVR). In this study, we investigated the potential of retinyl palmitate to enhance UVR‐induced photo‐co‐carcinogenesis. Groups of 36 male and 36 female SKH‐1 hairless mice were exposed to simulated solar light (SSL) and treated with the control cream or creams containing retinyl palmitate, 5 days per week for 40 weeks. Other groups of mice were exposed to SSL and received no cream treatment or received cream treatments and were exposed to ultraviolet‐A or ultraviolet‐B. Mice were monitored for the development of skin tumors, and the incidences and multiplicities of squamous cell neoplasia were determined by histopathology. In both the absence and presence of SSL, mice administered the control cream developed skin tumors earlier and had higher incidences and multiplicities of skin squamous cell neoplasms than mice that received no cream treatment. Compared to the control cream groups, mice exposed to SSL and administered the retinyl palmitate creams demonstrated earlier onsets of skin tumors and had increased incidences and multiplicities of squamous cell skin neoplasms.     

Noninvasive Optical Imaging of UV‐Induced Squamous Cell Carcinoma in Murine Skin: Studies of Early Tumor Development and Vitamin D Enhancement of Protoporphyrin IX Production

Better noninvasive techniques are needed to monitor protoporphyrin IX (PpIX) levels before and during photodynamic therapy (PDT) of squamous cell carcinoma (SCC) of the skin. Our aim was to evaluate (1) multispectral fluorescent imaging of ultraviolet light (UV)‐induced cancer and precancer in a mouse model of SCC and (2) multispectral imaging and probe‐based fluorescence detection as a tool to study vitamin D (VD) effects on aminolevulinic acid (ALA)‐induced PpIX synthesis. Dorsal skin of hairless mice was imaged weekly during a 24‐week UV carcinogenesis protocol. Hot spots of PpIX fluorescence were detectable by multispectral imaging beginning at 14 weeks of UV exposure. Many hot spots disappeared after cessation of UV at week 20, but others persisted or became visible after week 20, and corresponded to tumors that eventually became visible by eye. In SCC‐bearing mice pretreated with topical VD before ALA application, our optical techniques confirmed that VD preconditioning induces a tumor‐selective increase in PpIX levels. Fluorescence‐based optical imaging of PpIX is a promising tool for detecting early SCC lesions of the skin. Pretreatment with VD can increase the ability to detect early tumors, providing a potential new way to improve efficacy of ALA‐PDT.     

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.     

Hyperpigmentation induced by topical 5-aminolaevulinic acid plus visible light

Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an alternative tool for the treatment of superficial non-melanoma skin cancers. Recently ALA-PDT has been employed with encouraging results also for warts, condylomata and psoriasis. In this study the effects of topical ALA plus irradiation with visible light on intact human skin have been evaluated. Five skin areas (A, B, C, D, and E) on the inner upper part of the arms of five healthy volunteers (skin types III and IV) were treated with (A) ALA 20% in base cream without irradiation, (B) only the vehicle (base cream) without ALA, (C, D and E) ALA cream at the concentrations of 5, 10 and 20%, respectively; all treatments were applied with an occlusive dressing. Four hours after ALA or vehicle application areas B, C, D and E were irradiated with a fixed dose of 40 J/cm(2). ALA penetration through the intact skin was evaluated by in vivo fluorescence determination. The effects on healthy skin were evaluated by clinical and chromometric examinations, light microscopy, immunohistochemistry and electron microscopy. RESULTS: (1) in vivo fluorescence demonstrated that ALA is able to penetrate through the intact skin, when applied with occlusive dressing and induces a classical fluorescence peak due to Protoporphyrin IX (PpIX) formation, which is the active photosensitiser. (2) Skin areas receiving ALA plus irradiation showed erythema and swelling just after the irradiative session and hyperpigmentation 48-72 h later. (3) Colourimetric data confirmed significant skin colour changes: values a* (representing the erythematous changes) increased only on the skin areas where ALA+irradiation were applied and during the 48 h after irradiation, thereafter a* began to decrease; values L* (pigmentation) increased during the 2 weeks following treatment. (4) Histopathological, immunohistochemical (S100, HMB-45) and electron microscopic findings showed an absolute increment of the number of melanocytes, which appeared clearly activated. In conclusion the application of ALA cream followed by irradiation is able to induce a pigmentation response in healthy human skin, at least in skin types III and IV. This melanocytic activation could have a potential for the treatment of skin disorders characterised by hypopigmentation.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

HepG2 human hepatocarcinoma cells: an experimental model for photosensitization by endogenous porphyrins

Endogenous protoporphyurin IX (PpIX) synthesis after δ-aminolaevulinic acid (ALA) administration occurs in cancer cells in vivo; PpIX, which has a short half-life, may thus constitute a good alternative to haematoporphyrin derivative (HPD) (or Photofrin). This study assesses the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA, and compares ALA-induced toxicity and phototoxicity with the photodynamic therapy (PDT) effects of HPD on this cell line.

ALA induced a dose-dependent dark toxicity, with 79% and 66% cell survival for 50 and 100 μg ml⁻¹ ALA respectively after 3 h incubation; the same treatment, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), induced a dose-dependent phototoxicity, with 54% and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3 h delay before light exposure was found to be optimal to reach a maximum phototoxicity.

HPD induced a slight dose-dependent toxicity in HepG2 cells and a dose- and time-dependent phototoxicity ten times greater than that of ALA-PpIX PDT. After 3 h incubation of 2.5 and 5 μg ml⁻¹ HPD, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), cell survival was 59% and 24% respectively at 24 h.

Photoproducts induced by light irradiation of porphyrins absorb light in the red spectral region at longer wavelengths than the original porphyrins. The possible enhancement of PDT effects after HepG2 cell incubation with ALA or HPD was investigated by irradiating cells successively with red light (λ = 632 nm) and light (λ = 650 nm). The total fluence was kept constant at 25 J cm⁻². For both HPD and ALA-PpIX PDT, phototoxicity was lower when cells were irradiated for increased periods with λ = 650 nm light than with λ = 632 nm light alone. This suggests that any photoproducts involved either have a short life or are poorly photoreactive.

Not all cell lines can synthesize PpIX after ALA incubation. HepG2 cells, which can synthesize enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX PDT. In addition, ALA-PpIX PDT may represent a new, specific treatment for hepatocarcinomas.     

The Effects of Thrombocytopenia on Vessel Stasis and Macromolecular Leakage after Photodynamic Therapy Using Photofrin

Abstract— Several studies have reported thrombus formation and/or the release of specific vasoactive eicosanoids, suggesting that platelet activation or damage after photodynamic therapy (PDT) may contribute to blood flow stasis. The role of circulating platelets on blood flow stasis and vascular leakage of macromolecules during and after PDT was assessed in an intravital animal model. Sprague-Daw-ley rats bearing chondrosarcoma on the right hind limb were injected intravenously (i.v.) with 25 mg/kg Photofrin 24 h before light treatment of 135 J/cm² at 630 nm. Thrombocytopenia was induced in animals by administration of 3.75 mg/kg of rabbit anti-rat platelet antibody i.v. 30 min before the initiation of the light treatment. This regimen reduced circulating platelet levels from 300000/mm³ to 20000/mm³. Reductions in the luminal diameter of the microvasculature in normal muscle and tumor were observed in control animals given Photofrin and light. Venule leakage of macromolecules was noted shortly after the start of light treatment and continued throughout the period of observation. Animals made thrombocytopenic showed none of these changes after PDT in either normal tissues or tumor. The lack of vessel response correlated with the absence of thromboxane release in blood during PDT. These data suggest that platelets and eicosanoid release are necessary for vessel constriction and blood flow stasis after PDT using Photofrin.     

Induction of systemic neutrophil response in mice by photodynamic therapy of solid tumors.

Photodynamic therapy (PDT) of solid tumors elicits a strong, acute inflammatory response characterized by a rapid and massive infiltration of activated neutrophils into the tumor. The present study investigated the impact of PDT on the systemic and local (treatment site) kinetics of neutrophil trafficking and activity in mouse SCCVII and EMT6 tumor models. Differential leukocyte counts in the peripheral blood of treated mice revealed a pronounced neutrophilia developing rapidly after Photofrin porfimer sodium (Photofrin)- or tetra(m-tetrahydroxyphenyl)chlorin (mTHPC)-based PDT. Significant neutrophilia was also observed upon PDT treatment of normal dorsal skin but not on the footpad of tumor-free mice. The changes in circulating neutrophil numbers were accompanied by an efflux of these cells from the bone marrow. An increased proportion of cells with high L-selectin (CD62L antigen) expression was found among bone-marrow-residing neutrophils 6-24 h after PDT, and in neutrophils in the peripheral circulation and treated tumors 24 h after therapy. Complement inhibition completely prevented the development of PDT-induced neutrophilia. The results of the present study demonstrate that treatment of solid tumors by PDT induces a strong and protracted increase in systemic neutrophil numbers mediated by complement activation. This reaction reflects rapid and massive mobilization and activation of neutrophils for the destruction of PDT-treated tumor tissue.     

Topical bioadhesive patch systems enhance selectivity of protoporphyrin IX accumulation

In clinical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) of skin tumors it is desirable to develop vehicles that minimize the penetration of ALA through normal stratum corneum and maximize it through the compromised stratum corneum of the tumors to improve tumor selectivity. We have designed a bioadhesive patch, which may be able to achieve this aim. It induces levels of protoporphyrin IX (PpIX) in skin overlying tumors similar to those induced by the proprietary cream (Porphin) but at the same time induces less PpIX to form in normal skin and at distant sites. The mechanisms of action of the patch, as compared with that of the cream, were studied by means of Cuprophan barriers that mimic compromised tumor stratum corneum and in a mouse model with transplanted tumors.     

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mast cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132-135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2h found after ALA but not after MAL-PDT.     

Fluorescence imaging and phototoxicity effects of new formulation of chlorin e6-polyvinylpyrrolidone

Evaluations of the efficiency of a new formulation of chlorin consisting of a complex of trisodium salt chlorin e6 (Ce6) and polyvinylpyrrolidone (PVP) in photodynamic therapy (PDT) and fluorescence diagnosis was performed on poorly differentiated human bladder carcinoma murine model with the following specific aims: (i) to qualitatively evaluate the fluorescence accumulation in human bladder tumor, (ii) to determine fluorescence distribution of Ce6-PVP using the tissue extraction technique and fluorescence imaging technique, (iii) to compare the fluorescence distribution of Ce6, Ce6-PVP and Photofrin in skin of nude mice, and (iv) to investigate phototoxicity caused by different parameters (drug-light interval, drug dose, irradiation fluence rate and total light fluence) in PDT. The fluorescence of the Ce6-PVP formulation was determined either by fluorescence imaging measurements or by chemical extraction from the tissues displaying similar trends of distribution. Our results demonstrated that the Ce6-PVP formulation possesses less in vivo phototoxic effect compared to Ce6 alone. The phototoxicity revealed a strong dependence on the drug and light dosimetry as well as on the drug-light interval. In PDT, the Ce6-PVP compound was most toxic at the 1h drug-light interval at 200J/cm(2), while Ce6 alone was most toxic at a light dose of more that 50J/cm(2) at the 1 and 3h drug-light interval. We also confirmed that Ce6-PVP has a faster clearance compared to Ce6 alone or Photofrin. This eliminates the need for long-term photosensitivity precautions. In conclusion, the Ce6-PVP formulation seems to be a promising photosensitizer for fluorescence imaging as well as for photodynamic treatment.     

Fractionated Illumination at Low Fluence Rate Photodynamic Therapy in Mice

Photodynamic therapy (PDT) for actinic field cancerization is effective but painful. Pain mechanisms remain unclear but fluence rate has been shown to be a critical factor. Lower fluence rates also utilize available oxygen more efficiently. We investigated PDT effect in normal SKH1-HR mice using low and high fluence rate aminolevulinic acid (ALA) PDT and a fractionated illumination scheme. Six groups of six mice with different light treatment parameters were studied. Visual skin damage was assessed up to 7 days post-PDT. Fluorescence and reflectance spectroscopy during illuminations provided us with real-time information about protoporphyrin IX (PpIX) photobleaching. A novel dosing approach was introduced in that we used a photobleaching percentage instead of a preset fluence. Data show similar total and maximum damage scores in high and low fluence rate groups. Photobleaching of PpIX in the low fluence rate groups shows a trend toward more efficient photobleaching. Results indicate that low fluence rate PDT is as effective as and more efficient than high fluence rate PDT in normal mouse skin. Low fluence rate PDT light protocols need to be explored in human studies in search for an effective and well-tolerated treatment for actinic field cancerization.     

Combination of Oral Vitamin D3 with Photodynamic Therapy Enhances Tumor Cell Death in a Murine Model of Cutaneous Squamous Cell Carcinoma

Photodynamic therapy (PDT), in which 5‐ALA (a precursor for protoporphyrin IX, PpIX) is administered prior to exposure to light, is a nonscarring treatment for skin cancers. However, for deep tumors, ALA‐PDT is not always effective due to inadequate production of PpIX. We previously developed and reported a combination approach in which the active form of vitamin D₃ (calcitriol) is given systemically prior to PDT to improve PpIX accumulation and to enhance PDT‐induced tumor cell death; calcitriol, however, poses a risk of hypercalcemia. Here, we tested a possible strategy to circumvent the problem of hypercalcemia by substituting natural dietary vitamin D₃ (cholecalciferol; D₃) for calcitriol. Oral D₃ supplementation (10 days of a 10‐fold elevated D₃ diet) enhanced PpIX levels 3‐ to 4‐fold, and PDT‐mediated cell death 20‐fold, in subcutaneous A431 tumors. PpIX levels and cell viability in normal tissues were not affected. Hydroxylated metabolic forms of D₃ were only modestly elevated in serum, indicating minimal hypercalcemic risk. These results show that brief oral administration of cholecalciferol can serve as a safe neoadjuvant to ALA‐PDT. We suggest a clinical study, using oral vitamin D₃ prior to PDT, should be considered to evaluate this promising new approach to treating human skin cancer.     

Fluorescence Photobleaching of ALA-induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced dam-age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo-bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo-bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm², 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced dam-age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo-bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.     

Photodynamic therapy using 5-aminolaevulinic acid for oesophageal adenocarcinoma associated with Barrett's metaplasia

Photodynamic therapy (PDT) is a novel technique for local endoscopic treatment of gastrointestinal neoplasia. Current photosensitisers for PDT may cause prolonged skin phototoxicity. 5-Aminolaevulinic acid (ALA), a precursor of the photosensitiser protoporphyrin IX (PpIX), is more acceptable because of its short half-life and preferential accumulation in mucosa and mucosal tumour. We have treated 12 patients, median age 73 years (range 55-88) with oesophageal adenocarcinoma arising from Barrett's metaplasia (two carcinomas-in-situ, grade 0; 10 carcinomas, grade 1-11A based on endoluminal ultrasound in two and CT scanning in 10 patients). ALA (60 and 75 mg/kg body weight) was given orally in two or five equally divided doses. The PpIX distribution in stomach, normal oesophagus, Barrett's mucosa and carcinoma was measured by quantitative fluorescence photometry. PDT was performed using laser light (630 nm) delivered via a cylindrical diffuser 4-6 h after the first dose of ALA. The patients received one to four sessions of PDT. PpIX accumulation in the mucosa was two to three times that in the lamina propria. The differential distribution between carcinomatous and normal oesophageal mucosa was less marked (carcinoma:normal mucosa ratio = 1.4). Higher doses of ALA increased PpIX accumulation in all tissues but did not increase the differential PpIX distribution between tumour and normal oesophageal mucosa. After PDT using ALA (ALA/PDT), all mucosa showed superficial white necrotic changes and the histology confirmed fibrinoid necrosis. One patient with carcinoma-in-situ had the tumour eradicated after one treatment with no recurrence at 28 months. Another patient with a small T1 tumour required four ALA/PDT treatments, and died of other disease after 36 months. There was no evidence of recurrence. The tumour bulk in the other carcinomas was not significantly reduced. ALA/PDT has a potential for the eradication of small tumours but careful patient selection with endoluminal ultrasound is needed when using ALA/PDT to treat oesophageal cancer.