Topical Photodynamic Therapy Using Different Porphyrin Precursors Leads to Differences in Vascular Photosensitization and Vascular Damage in Normal Mouse Skin

Different distributions of hexyl aminolevulinate (HAL), aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) in the superficial vasculature are not well studied but they are hypothesized to play an important role in topical photodynamic therapy (PDT). The colocalization of fluorescent CD31 and protoporphyrin IX (PpIX) was calculated using confocal microscopy of mouse skin sections to investigate the vascular distribution after topical application. Vascular damage leads to disruption of the normal endothelial adherens junction complex, of which CD144 is an integral component. Therefore, normal CD31 combined with loss of normal fluorescent CD144 staining was visually scored to assess vascular damage. Both the vascular PpIX concentration and the vascular damage were highest for HAL, then ALA and then MAL. Vascular damage in MAL was not different from normal contralateral control skin. This pattern is consistent with literature data on vasoconstriction after PDT, and with the hypothesis that the vasculature plays a role in light fractionation that increases efficacy for HAL and ALA‐PDT but not for MAL. These findings indicate that endothelial cells of superficial blood vessels synthesize biologically relevant PpIX concentrations, leading to vascular damage. Such vascular effects are expected to influence the oxygenation of tissue after PDT which can be important for treatment efficacy.     

5-Aminolevulinic Acid Photosensitization of Dysplastic Barrett's Esophagus: A Pharmacokinetic Study

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) may have a role in the treatment of dysplastic Barrett's esophagus. Before ALA-induced PDT can be used clinically, optimum treatment parameters must be established. In this study of 35 patients, the issues of drug dosage, time interval between drug and light delivery and side effects of oral ALA administration are addressed. Spectrofluorometric analysis of tissue samples demonstrates that oral ALA administration induces porphyrin accumulation in esophageal tissues, with maximum levels at 4-6 h. High-performance liquid chromatography confirms the identity of this porphyrin as PpIX, and fluorescence microscopy analysis demonstrates that it preferentially accumulates in the esophageal mucosa, rather than in the underlying stroma. Side effects of ALA administration included malaise, headache, photosensitivity, alopecia, transient derangement of liver function, nausea and vomiting. Fewer side effects and less hepatic toxicity was seen with 30 mg/kg than 50 mg/kg ALA. In conclusion, oral ALA administration induces preferential PpIX accumulation in the esophageal mucosa, with peak PpIX fluorescence noted at 4 h and minimal systemic toxicity at a dose of 30 mg/kg.     

Systemic application of photosensitizers in the chick chorioallantoic membrane (CAM) model: photodynamic response of CAM vessels and 5-aminolevulinic acid uptake kinetics by transplantable tumors.

The aim of this study is to modify the chick chorioallantoic membrane (CAM) model into a whole-animal tumor model for photodynamic therapy (PDT). By using intraperitoneal (i.p.) photosensitizer injection of the chick embryo, use of the CAM for PDT has been extended to include systemic delivery as well as topical application of photosensitizers. The model has been tested for its capability to mimic an animal tumor model and to serve for PDT studies by measuring drug fluorescence and PDT-induced effects. Three second-generation photosensitizers have been tested for their ability to produce photodynamic response in the chick embryo/CAM system when delivered by i.p. injection: 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA), and Lutetium-texaphyrin (Lu-Tex). Exposure of the CAM vasculature to the appropriate laser light results in light-dose-dependent vascular damage with all three compounds. Localization of ALA following i.p. injections in embryos, whose CAMs have been implanted with rat ovarian cancer cells to produce nodules, is determined in real time by fluorescence of the photoactive metabolite protoporphyrin IX (PpIX). Dose-dependent fluorescence in the normal CAM vasculature and the tumor implants confirms the uptake of ALA from the peritoneum, systemic circulation of the drug, and its conversion to PpIX.     

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.     

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.     

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.     

Drug delivery of aminolevulinic acid from topical formulations intended for photodynamic therapy

Topical photodynamic therapy is used for a variety of malignant and pre-malignant skin disorders, including Bowen's Disease and Superficial Basal Cell Carcinoma. A haem precursor, typically 5-aminolevulinic acid (ALA), acting as a prodrug, is absorbed and converted by the haem biosynthetic pathway to photoactive protoprophyrin IX (PpIX), which accumulates preferentially in rapidly dividing cells. Cell destruction occurs when PpIX is activated by an intense light source of appropriate wavelength. Topical delivery of ALA avoids the prolonged photosensitivity reactions associated with systemic administration of photosensitisers but its clinical utility is influenced by the tissue penetration characteristics of the drug, its ease of application and the stability of the active agent in the applied dose. This review, therefore, focuses on drug delivery applications for topical, ALA-based PDT. Issues considered in detail include physical and chemical enhancement strategies for tissue penetration of ALA and subsequent intracellular accumulation of PpIX, together with formulation strategies and drug delivery design solutions appropriate to various clinical applications. The fundamental aspects of drug diffusion in relation to the physicochemical properties of ALA are reviewed and specific consideration is given to the degradation pathways of ALA in formulated systems that, in turn, influence the design of stable topical formulations.     

Kinetic fluorescence studies of 5-aminolaevulinic acid-induced protoporphyrin IX accumulation in basal cell carcinomas.

Laser-induced fluorescence (LIF) investigations have been performed in connection with photodynamic therapy (PDT) of basal cell carcinomas and adjacent normal skin following topical application of 5-aminolaevulinic acid (ALA) in order to study the kinetics of the protoporphyrin IX (PpIX) build-up. Five superficial and 10 nodular lesions in 15 patients are included in the study. Fluorescence measurements are performed prior to the application of ALA, 2, 4 and 6 h post ALA application, immediately post PDT (60 J cm-2 at 635 nm), and 2 h after the treatment. Hence, the build-up, photobleaching and re-accumulation of PpIX can be followed. Superficial lesions show a maximum PpIX fluorescence 6 h post ALA application, whereas the intensity is already the highest 2-4 h after the application in nodular lesions. Immediately post PDT, the fluorescence contribution at 670 nm from the photoproducts is about 2% of the pre-PDT PpIX fluorescence at 635 nm. Two hours after the treatment, a uniform distribution of PpIX is found in the lesion and surrounding normal tissue. During the whole procedure, the autofluorescence of the lesions and the normal skin does not vary significantly from the values recorded before the application of ALA.     

Detection of Early Stages of Carcinogenesis in Adenomas of Murine Lung by 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence

Abstract— Administration of the heme precursor 5-aminolevulinic acid (ALA) leads to the selective accumulation of the photosensitizer protoporphyrin IX (PpIX) in certain types of normal and abnormal tissues. This phenomenon has been exploited clinically for detection and treatment of a variety of malignant and nonmalignant lesions. The present preclinical study examined the specificity of ALA-induced porphyrin fluorescence in chemically induced murine lung tumors in vivo. During the early stages of tumorigenesis, ALA-induced PpIX fluorescence developed in hyperplastic tissues in the lung and later in early lung tumor foci. In early tumor foci, maximum PpIX fluorescence occurred 2 h after the administration of ALA and returned to background levels after 4 h. There was approximately a 20-fold difference in PpIX fluorescence intensity between tumor foci and the adjacent normal tissue. The specificity of ALA-induced fluorescence for hyperplastic tissues and benign tumors in lung during tumorigenesis suggests a possible use for this fluorochrome in the detection of premalignant alterations in the lung by fluorescence endoscopy. Two non-small cell lung cancer cell lines developed ALA-induced PpIX fluorescence in vitro . These lines exhibited a light-dose-dependent phototoxic response to ALA photodynamic therapy (PDT) in vitro . Because PpIX is a clinically effective photosensitizer for a wide variety of malignancies, these results support the possible use of ALA-induced PpIX PDT for lung cancer.     

Oral aminolevulinic acid induces protoporphyrin IX fluorescence in psoriatic plaques and peripheral blood cells

Photodynamic therapy (PDT) with topical aminolevulinic acid (ALA) has been shown in previous studies to improve psoriasis. However, topical ALA-PDT may not be practical for the treatment of extensive disease. In order to overcome this limitation we have explored the potential use of oral ALA administration in psoriatic patients. Twelve patients with plaque psoriasis received a single oral ALA dose of 10, 20 or 30 mg/kg followed by measurement of protoporphyrin IX (PpIX) fluorescence in the skin and circulating blood cells. Skin PpIX levels were determined over time after ALA administration by the quantification of the 635 nm PpIX emission peak with in vivo fluorescence spectroscopy under 442 nm laser excitation. Administration of ALA at 20 and 30 mg/kg induced preferential accumulation of PpIX in psoriatic as opposed to adjacent normal skin. Peak fluorescence intensity in psoriatic and normal skin occurred between 3 and 5 h after the administration of 20 and 30 mg/kg, respectively. Ratios of up to 10 for PpIX fluorescence between psoriatic versus normal skin were obtained at the 30 mg/kg dose of ALA. Visible PpIX fluorescence was also observed on normal facial skin, and nonspecific skin photosensitivity occurred only in patients who received the 20 or 30 mg/kg doses. PpIX fluorescence intensity was measured in circulating blood cells by flow cytometry. PpIX fluorescence was higher in monocytes and neutrophils as compared to CD4+ and CD8+ T lymphocytes. PpIX levels in these cells were higher in patients who received higher ALA doses and peaked between 4 and 8 h after administration of ALA. There was only a modest increase in PpIX levels in circulating CD4+ and CD8+ T lymphocytes. In conclusion oral administration of ALA induced preferential accumulation of PpIX in psoriatic plaques as compared to adjacent normal skin suggesting that PDT with oral ALA should be further explored for the treatment of psoriasis.     

Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.     

Kinetics of Photobleaching of Protoporphyrin IX in the Skin of Nude Mice Exposed to Different Fluence Rates of Red Light

Abstract— The purpose of the present study was to determine the kinetics and the fluence rate dependency of the photo-bleaching of protoporphyrin IX (PpIX) in normal skin of Balb/c nude mice after systemic and topical application of 5-aminolevulinic acid (ALA). ALA was administered systemically (200 mg/kg body weight, i.p.) and topically (20% w/w ALA cream) to the mice. Fluences of up to 40 J/cm² were delivered by a dye laser (636 nm) at fluence rates of 37.5, 75, 150, 300 and 500 mW/cm². The photo-bleaching rate was constant within this range of fluence rates. This result suggests that there is no oxygen effect for PpIX photobleaching in this region for the skin of Balb/c nude mice. During light exposure the fluorescence decay followed neither first- nor second-order kinetics. The decay rate was slightly faster after systemic application than after topical application of ALA, but did not depend on the time (1–8 h) between application and analysis.     

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.     

Selective distribution of porphyrins in skin thick basal cell carcinoma after topical application of methyl 5-aminolevulinate

Topical photodynamic therapy (PDT) of superficial basal cell carcinoma (BCC) with 5-aminolevulinic acid (ALA) has achieved promising clinical results. However, the efficacy of this therapy for thick BCC is dramatically decreased by a limited diffusion of hydrophilic ALA into the tumor. Lipophilic esters of ALA may enhance their penetration into the lesion. In this randomized, open clinical study, microscopic fluorescence photometry incorporating a light-sensitive thermo-electrically cooled charge-coupled device (CCD) camera was employed to investigate the penetration of methyl 5-aminolevulinate-induced porphyrin fluorescence in thick BCC lesions. Both the distribution pattern and the amount of porphyrins in 32 lesions of 16 patients were studied after topical application of 16, 80 or 160 mg/g of methyl 5-aminolevulinate for 3 or 18 h. A highly selective and homogeneous distribution of methyl 5-aminolevulinate-induced porphyrin fluorescence was seen in all lesions studied, with much less fluorescence in the adjacent normal skin tissues. In lesions of up to 2 mm thickness the application of 160 mg/g methyl 5-aminolevulinate for 3 h showed the highest ratio of porphyrin fluorescence depth to tumor depth (0.98+/-0.04), thus providing a biologic rationale for a clinical PDT trial with this regimen.     

The Hydroxypyridinone Iron Chelator CP94 Can Enhance PpIX-induced PDT of Cultured Human Glioma Cells

Photodynamic therapy (PDT) with the pro-drugs 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. To potentially increase accumulation of the photosensitizer, protoporphyrin IX (PpIX), within tumor cells an iron chelator can be employed. This study analyzed the effects of ALA/MAL-induced PDT combined with the iron chelator 1, 2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) on the accumulation of PpIX in human glioma cells in vitro. Cells were incubated for 0, 3 and 6 h with various concentrations of ALA/MAL with or without CP94 and the resulting accumulations of PpIX, which naturally fluoresces, were quantified prior to and following light irradiation. In addition, counts of viable cells were recorded. The use of CP94 in combination with ALA/MAL produced significant enhancements of PpIX fluorescence in human glioma cells. At the highest concentrations of each prodrug, CP94 enhanced PpIX fluorescence significantly at 3 h for ALA and by more than 50% at 6 h for MAL. Cells subsequently treated with ALA/MAL-induced PDT in combination with CP94 produced the greatest cytotoxicity. It is therefore concluded that with further study CP94 may be a useful adjuvant to photodiagnosis and/or PpIX-induced PDT treatment of glioma.     

Fractionated illumination in oesophageal ALA-PDT: effect on ferrochelatase activity

BACKGROUND AND OBJECTIVE: Administration of 5-aminolevulinic acid (ALA) induces accumulation of the photosensitive compound protoporphyrin IX (PpIX) in certain tissues. PplX can be used as photosensitizer in photodynamic therapy (PDT). More selective or higher PpIX accumulation in the area to be treated could optimize the results of ALA-PDT. Porphobilinogen deaminase (PBGD) is rate-limiting in PpIX formation whereas ferrochelatase converts PpIX into haem by chelation of ferrous iron into PpIX. This results in a moment of close interaction (ferrochelatase binding to PpIX) during which ferrochelatase could selectively be destroyed resulting in an increased PpIX concentration. The aim of the present study was to investigate whether illumination before PDT can selectively destroy ferrochelatase. and whether this results in higher PpIX accumulation and thereby increases the PDT effect. Furthermore, the effect of a second ALA dose was tested. STUDY DESIGN/MATERIALS AND METHODS: Oesophageal tissue of 60 rats were allocated to 2 groups of 30 animals each. In one group, enzyme and PpIX measurements were performed after ALA administration (200 mg/kg orally, n=20), or a second dose of 200 mg/kg ALA at 4 h (n=10), half of each group with and without illumination at 1 h with 12.5 J/cm diffuser length. In the second group, PDT was performed. Ten animals were illuminated at 3 h after ALA administration with 20 (n=5) or 32.5 J/cm (n=5), 10 animals were illuminated at 1 h (12.5 J/cm) and received intra-oesophageal PDT treatment (20 J/cm) at 3 h (n=5) or 4 h (n=5) after ALA. Additionally, 10 animals received a second dose of 200 mg/kg ALA at 4 h and were illuminated (20 J/cm) at 7 h after the first dose of ALA with (n=5) or without (n=5) illumination at 4 h (12.5 J/cm). RESULTS: Illumination with 12.5 J/cm at 1 h after ALA administration caused inhibition of the activity of ferrochelatase at 3 and 4 h after ALA (P=0.02 and P<0.001, respectively), but not at 7 h (P=0.3). In animals sacrificed at 4 h the ratio PBGD:ferrochelatase was higher in animals illuminated at 1 h compared to non-illuminated animals (P<0.001). PpIX concentration was highest (42.7 +/- 3.2 pmol/mg protein) at 3 h after ALA administration and did not increase by illumination at 1 h. Administration of a second dose of ALA did not result in higher PpIX accumulation. After PDT, no difference in epithelial or muscular damage was found between the various groups. CONCLUSION: Illumination at 1 h after ALA administration can cause selective destruction of ferrochelatase, resulting in a higher ratio of PBGD:ferrochelatase. This does not result in accumulation of more porphyrins, even when a second dose of ALA is given. Therefore, under the conditions used in this study fractionated illumination does not enhance ALA-PDT-induced epithelial ablation of the rat oesophagus.     

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.     

5‐Aminolevulinic acid‐Loaded Fullerene Nanoparticles for In Vitro and In Vivo Photodynamic Therapy

This report explores some properties of 80–200 nm nanoparticles containing 5‐aminolevulinic acid (ALA) and fullerene (C60) for photodynamic therapy (PDT). Compared with ALA, the nanoparticles yielded more protoporphyrin IX (PpIX) formation in cells and tissues and to a significant improvement in antitumor efficacy in tumor‐bearing mice. Maximum levels of PpIX were obtained 4 h after administration and selective PpIX formation in tumor was observed. These nanoparticles appear to be a useful vehicle for drug delivery purposes. In this study, a procedure for preparing fullerene nanoparticles containing ALA was developed. The product alone exhibited no detectable toxicity in the dark and was superior to ALA alone in promoting PpIX biosynthesis and PDT efficacy both in culture and in a murine tumor model. These results suggest that this procedure could be the basis for an improved PDT protocol for cancer control.     

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.     

An in vitro comparison of the effects of the iron-chelating agents, CP94 and dexrazoxane, on protoporphyrin IX accumulation for photodynamic therapy and/or fluorescence guided resection

Photodynamic therapy (PDT) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. Maximizing the accumulation of the photosensitizer protoporphyrin IX (PpIX) within different cell types would be clinically useful. Dermatological PpIX-induced PDT regimes produce good clinical outcomes but this currently only applies when the lesion remains superficial. Also, as an adjuvant therapy for the treatment of primary brain tumors, fluorescence guided resection (FGR) and PDT can be used to highlight and destroy tumor cells unreachable by surgical resection. By employing iron chelators PpIX accumulation can be enhanced. Two iron-chelating agents, 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) and dexrazoxane, were individually combined with the porphyrin precursors aminolevulinic acid (ALA), methyl aminolevulinate (MAL) and hexyl aminolevulinate (HAL). Efficacies of the iron-chelating agents were compared by recording the PpIX fluorescence in human squamous epithelial carcinoma cells (A431) and human glioma cells (U-87 MG) every hour for up to 6 h. Coincubation of ALA/MAL/HAL with CP94 resulted in a greater accumulation of PpIX compared to that produced by coincubation of these congeners with dexrazoxane. Therefore the clinical employment of iron chelation, particularly with CP94 could potentially increase and/or accelerate the accumulation of ALA/MAL/HAL-induced PpIX for PDT or FGR.