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.     

5-Aminolevulinic acid-induced protoporphyrin-IX accumulation and associated phototoxicity in macrophages and oral cancer cell lines

Studies were carried out on 5-aminolevulinic acid (ALA)-induced protoporphyrin (PpIX) synthesis in mice peritoneal macrophages and two human oral squamous cell carcinoma (OSCC) cell lines NT8e and 4451. Cells were treated with 200 microg/ml ALA for 15 h and PpIX accumulation was monitored by spectrofluorometry and phototoxicity to red light (630+/-20 nm) was measured by MTT assay. PpIX accumulation was higher in macrophages as compared to OSCC cells under both normal serum concentration (10%) and conditions of serum depletion. The results on phototoxicity measurements correlated well with the levels of PpIX accumulation in both macrophages and cancer cells. While red light caused 20% phototoxicity in macrophages, no phototoxicity was seen in 4451 cells at 10% serum. Decrease in serum concentration to 5% and 1% led to higher phototoxicity corresponding to 40% and 70% in macrophages and 10% and 15% in 4451 cells. Similar results were obtained in NT8e cell line. Propidium iodide staining followed by fluorescence microscopic observations on photodynamically treated co-culture of murine or human macrophages and cancer cells showed selective damage to macrophages. These results suggest that in OSCC, macrophages would contribute more to tumor PpIX level than tumor cells themselves and PDT may lead to selective killing of macrophages at the site of treatment. Since macrophages are responsible for production and secretion of various tumor growth mediators, the effect of selective macrophage killing on the outcome of PDT would be significant.     

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.     

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.     

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.     

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.     

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.     

The influence of UV exposure on 5-aminolevulinic acid-induced protoporphyrin IX production in skin.

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).     

Protoporphyrin IX fluorescence kinetics and localization after topical application of ALA pentyl ester and ALA on hairless mouse skin with UVB-induced early skin cancer

In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.     

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.     

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.     

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insuffici?nt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.     

Fluorescence kinetics of protoporphyrin-IX induced from 5-ALA compounds in rabbit postballoon injury model for ALA-photoangioplasty

Abstract Protoporphyrin IX (PpIX) is one of the photodynamically active substances that are endogenously synthesized in the metabolic pathway for heme as a precursor. Aminolevulinic acid-esters are more lipophilic than conventional 5-aminolevulinic acid (ALA) and some of them are currently being approved as new drugs for photodynamic diagnosis (PDD) and photodynamic therapy (PDT). In order to investigate the pharmacokinetics of ALA and ALA-ethyl ester (ALA-ethyl) in the atheromatous plaque and normal aortic wall of rabbit postballoon injured artery, each 60 mg kg(-1) of ALA or ALA-ethyl was injected intravenously followed by serial detection of PpIX fluorescence of harvested arteries at 0-48 h post-injection. Maximum PpIX build-up in the atheromatous plaque was seen at 2 h after injecting ALA. In contrast, it occurred at 9 h after injecting ALA-ethyl. In addition, the selective build-up of ALA in the atheromatous plaque compared to normal vessel wall was much higher (10 times) than that of ALA-ethyl. The time of maximum fluorescence intensity of PpIX was employed as drug-light-interval for subsequent PDT treatment of the atheromatous plaque with 50-150 J cm(-1) of light dose. Significant reduction in plaque was observed without damage of the medial wall at both groups, but smooth muscle cell (SMC) was still present in the media region below the PDT-treated atheromatous plaque. In conclusion, ALA may be a more effective compound for endovascular PDT treatment of the atheromatous plaque compared with ALA-ethyl based on their pharmacokinetics, but further optimization of PDT methodology remains to remove completely residual SMC in the media for preventing potential restenosis.     

Dose and timing of the first light fraction in two-fold illumination schemes for topical ALA-mediated photodynamic therapy of hairless mouse skin

A fractionated illumination scheme in which a cumulative fluence of 100 J cm(-2) is delivered in two equal light fractions separated by a dark interval of 2 h has been shown to considerably increase the efficacy of 5-aminolevulinic acid (ALA)-photodynamic therapy (PDT). The efficacy of such a scheme is further increased if the fluence of the first light fraction is reduced to 5 J cm(-2). We have investigated the relationship between the PDT response and the kinetics of protoporphyrin IX (PpIX) fluorescence in the SKH1 HR hairless mouse for first fraction fluences below 5 J cm(-2) delivered 4 h after the application of ALA and 10 J cm(-2) delivered 2 h after the application of ALA. Illumination is performed using 514 nm at a fluence rate of 50 mW cm(-2). Reducing the fluence of the first fraction to 2.5 J cm(-2) does not result in significantly different visual skin damage. The PDT response, however, is significantly reduced if the fluence is lowered to 1 J cm(-2), but this illumination scheme (1 + 99 J cm(-2)) remains significantly more effective than a single illumination of 100 J cm(-2). A first light fraction of 10 J cm(-2) can be delivered 2 h earlier, 2 h after the application of ALA, without significant reduction in the PDT response compared with 5 + 95 J cm(-2) delivered 4 and 6 h after the application of ALA. The kinetics of PpIX fluorescence are consistent with those reported previously by us and do not explain the significant increase in PDT response with a two-fold illumination scheme. Histological sections of the illuminated volume showed a trend toward increasing extent and depth of necrosis for the two-fold illumination scheme in which the first light fraction is 5 J cm(-2), compared with a single illumination scheme.     

Effects of Silencing Heme Biosynthesis Enzymes on 5‐Aminolevulinic Acid‐mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy

Aminolevulinic acid (ALA)‐mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA‐mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA‐mediated PDT. Silencing PBGS or PBGD significantly reduced ALA‐stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA‐stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA‐stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA‐PDT, while increased sensitivity to ALA‐PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA‐mediated PpIX fluorescence and PDT efficacy.     

Topical 5-aminolevulinic acid-photodynamic therapy of hairless mouse skin using two-fold illumination schemes: PpIX fluorescence kinetics, photobleaching and biological effect

Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm-2 was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm-2. In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 + 95 and 50 + 50 J cm-2 (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm-2 in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm-2 is delivered in the first light fraction. Also, delivering 5 J cm-2 at 5 mW cm-2 + 95 J cm-2 at 50 mW cm-2 results in a reduction in visual skin damage from that obtained with 5 + 95 J cm-2 at 50 mW cm-2. A similar reduction in damage is observed if 5 + 45 J cm-2 are delivered at 50 mW cm-2. PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.     

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.     

5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.     

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.     

Quantification of topically delivered 5-aminolevulinic acid by lontophoresis across ex vivo human stratum corneum

Iontophoretic transport of the prodrug 5-aminolevulinic acid (ALA), which is used for photodynamic therapy (PDT), across human stratum corneum (SC) was studied quantitatively in vitro. The experiments were carried out in a three-compartment iontophoresis cell consisting of two electrode chambers equipped with Ag-AgCl electrodes, each separated from a central acceptor chamber by a sheet of SC, supported by a dialysis membrane, to mimic the side-by-side configuration normally used in vivo. Acceptor fluid samples were collected every hour for a period of 30 h in a fraction collector and analyzed by high-performance liquid chromatography-fluorometry after derivatization of the ALA. The iontophoretic ALA flux was studied as a function of the applied current density and the ALA concentration in the donor solution (1, 2.5 or 10% ALA). Depending on the ALA concentration in the donor cell, iontophoresis enhances the flux from close to the detection limit of 0.23 nmol cm(-2) h(-1) at zero current density (passive diffusion) to several hundred or thousand nanomoles per square centimeter per hour at current densities ranging from 100 to 1000 microA cm(-2). For example, interpolating our data we find that with an ALA concentration of 2% in the donor chamber, a current density of 0.255 mA cm(-2) transports 0.065 micromol cm(-2) ALA across the SC in 10 min (conditions of Rhodes et al. (1997), J. Invest. Dermatol. 108, 87-91). For passive diffusion we find that a 5 h topical application of 20% ALA results in the transport of 0.05 micromol cm(-2). Thus, the amount of ALA that passively diffuses through the SC in several hours, leading to therapeutic levels of protoporphyrin IX (PpIX) in the epidermis, can be delivered by iontophoresis in 10 min or less. However, because the formation of sufficient PpIX also requires several hours and also because the SC overlying skin lesions such as basal cell carcinoma (BCC) is not intact, the clinical benefit of topical ALA delivery by iontophoresis for PDT of BCC is yet to be established.