The 5-aminolevulinic acid-induced porphyrin biosynthesis in benign and malignant cells of the skin.

In fluorescence diagnosis and photodynamic therapy of neoplastic tissues 5-aminolevulinic acid is used to synthesize endogenous porphyrins as photosensitizers. The efficacy of neoplastic tissues to fluorescence diagnosis and photodynamic therapy is thought to be dependent on the total level of intralesional formed porphyrins. The available profiles of porphyrin metabolites in normal and in neoplastic cell lines after administration of 5-aminolevulinic acid vary considerably. Thus, this is the first in-vitro study which compares the porphyrin biosynthesis in normal skin cells (HaCaT, fibroblasts) with melanoma cells (Bro, SKMel-23, SKMel-28). After incubation with 1 mM 5-aminolevulinic acid, kinetics of porphyrin levels and metabolites were determined in the cells and the corresponding supernatants. Exogenous 5-aminolevulinic acid induced porphyrin formation in all cells with maximum values after an incubation period of 16-36 h. Increase of porphyrin levels varied from 10- to 80-fold (SKMel-28>HaCaT>fibroblasts>SKMel-23>Bro) with minimum 1.5 times higher levels of porphyrins in the supernatants than in the cells. In cells and supernatants protoporphyrin and coproporphyrin were the predominantly formed porphyrin metabolites. Metastatic melanoma cells (SKMel-23, SKMel-28) accumulated much higher porphyrin levels than primary melanoma cells (Bro). In conclusion, by optimizing the treatment modalities, especially the light source, topical photodynamic therapy (PDT) could become a treatment alternative of melanoma metastases in progressive disease.     

Preferential Relative Porphyrin Enrichment in Solar Keratoses upon Topical Application of ^-Aminolevulinic Acid Methylester

Topically applied δ-aminolevulinic acid is used efficiently for the treatment of solar keratoses by photodynamic therapy. Recent animal studies suggest that porphyrin sensitization of epithelial tissue is improved by using esters rather than free δ-aminolevulinic acid. The present study examines porphyrin metabolite formation after topical application of δ-aminolevulinic acid or δ-aminolevulinic acid methylester in human solar keratoses versus adjacent normal skin. Levels of total porphyrins, porphyrin metabolites and protein were measured in skin samples excised after 1 and 6 h. Higher levels of porphyrins were observed in solar keratoses than in normal skin with both substances. Maximum porphyrin levels were present in solar keratoses treated with δ-aminolevulinic acid for 6 h. However, the ratio of porphyrins in solar keratoses versus adjacent normal skin was higher with 8-aminolevulinic acid methylester. The pattern of porphyrins showed no significant difference between normal and afflicted skin, protoporphyrin being predominant. The results suggest that application of free δ-aminolevulinic acid may be more effective in sensitizing solar keratoses. However, treatment with 8-aminolevulinic acid methylester leads to a preferential enrichment of porphyrins within lesional skin.     

Inactivation of erythrocytic, lymphocytic and myelocytic leukemic cells by photoexcitation of endogenous porphyrins

The photodynamic sensitization of leukemic cells (erythrocytic, myelocytic and lymphocytic) via light activation of endogenous porphyrins is described. Human myelocytic-erythrocytic K562 cells and murine Friend erythroleukemia (FELC) and T-cell lymphoma Eb-Esb cells were stimulated to synthesize and accumulate porphyrins. K562 cells accumulated high amounts of protoporphyrin by stimulation with 5-aminolevulinic acid (ALA) plus sodium butyrate or hemin. For Friend and Eb-Ebs cells ALA was an adequate stimulator. The high-metastatic Esb lymphoma cells accumulated comparatively more porphyrin than the low-metastatic Eb cell line. Maximal porphyrin accumulation produced mortality rates of more than 99% after 10 min of photoactivation of the three leukemic lines. Thymidine incorporation was inhibited by the photodynamic effect depending on porphyrin concentration. These results confirm the photodynamic ability of endogenous porphyrins to inactivate cancer cells of different origins.     

PROTOPORPHYRIN-INDUCED PHOTODAMAGE: STUDIES USING CULTURED SKIN FIBROBLASTS

A new system is described for the study of protoporphyrin-induced photodamage of cells. This system differs from those previously described in that fibroblasts are induced to synthesize protoporphyrin from its precursor δ-aminolevulinic acid.
Fibroblasts were cultured from skin biopsies of 6 normal individuals and 6 patients with protoporphyria. All cell lines in both groups accumulated protoporphyrin when incubated with δ-aminolevulinic acid in the absence of iron. Irradiation for 2 min with long-wave UV light caused death of cells which had accumulated protoporphyrin, but not of cells which had been incubated without δ-aminolevulinic acid. Cell damage could be quantitatively assessed by the release of chromium-51 into the medium.
Examination of irradiated protoporphyrin-rich fibroblasts by electron microscopy revealed no significant differences between lines from patients with protoporphyria and normal individuals. The earliest indications of photodamage were rarifaction of the mitochondrial matrix with dilatation of cristae. dilatation of endoplasmic reticulum, and loss of plasma membrance integrity. Condensation of the cells correlated with cell death. These structural alterations suggest a generalized cellular injury.     

NON-INVASIVE TECHNIQUE FOR OBTAINING FLUORESCENCE EXCITATION AND EMISSION SPECTRA IN VIVO

An intensified photodiode array forms the heart of a sensitive spectrophotofluorometry system that permits the rapid and non-invasive determination of fluorescence emission spectra in the skin of living, non-anesthetized animals. Using this system, we found it possible to obtain good emission and excitation spectra of the material responsible for the weak red fluorescence that characterizes normal mouse skin, and to follow the biosynthesis and subsequent clearance of protoporphyrin IX in the skin of non-anesthetized mice that had been given various doses of the porphyrin precursor 5-aminolevulinic acid.     

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.     

INACTIVATION BY MONOCHROMATIC NEAR-UV RADIATION OF AN Escherichia coli hemA8 MUTANT GROWN WITH AND WITHOUT δ-AMINOLEVULINIC ACID: THE ROLE OF DNA vs MEMBRANE DAMAGE

Abstract— Escherichia coli strain RT8 hemA8 [blocked in biosynthesis of δ-aminolevulinic acid (δ-ALA), and unable to manufacture porphyrins unless exogenously supplied with δ-ALA] is inactivated more efficiently by monochromatic 334- and 405-nm radiations if the cells are grown with δ-ALA supplementation. The fiuence enhancement factor for δ-ALA sensitization is larger for light at 405 nm than at 334 nm. Both irradiation conditions (plus or minus δ-ALA) showed prominent oxygen enhancement ratios, which were also larger at 405 nm than at 334 nm. At 334 nm, δ-ALA supplementation did not affect the accumulation of DNA breaks, while at 405 nm, the induction of DNA breaks doubled for cells supplemented with δ-ALA. Rubidium leakage caused by 405-nm radiation occurred at a smaller fiuence in cells supplemented with higher concentrations of δ-ALA than in cells supplemented with a lower concentration. The results suggest that (1) porphyrin derivatives may have a role in cell killing by near-UV radiations, and (2) damage to cytomembranes may be a critical lesion produced by these events, whereas DNA breakage may not.     

Targeting of sebocytes by aminolevulinic acid-dependent photosensitization

Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX has been developed as a very useful therapeutic modality. Recently, several authors have reported on the efficacy of this procedure for acne. This approach is based on the fact that 5-aminolevulinic acid-induced protoporphyrin IX has strong selectivity for sebaceous glands. We used the immortalized human sebaceous gland cell line SZ95 to investigate cellular mechanisms of photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX. Quantification of induced protoporphyrin IX production showed dependence on the applied 5-aminolevulinic acid dose. When SZ95 sebocytes were differentiated by arachidonic acid treatment, there was no difference between them and the control cells with respect to both the amount of 5-aminolevulinic acid-induced protoporphyrin IX and the phototoxic effects. We altered protoporphyrin IX formation rates by growing cells scattered as single cells in the culture dishes. Single cells produced significantly lower protoporphyrin IX levels than those grown with intercellular contacts. Intracellular localization of protoporphyrin IX was imaged using confocal laser scanning microscopy. The differentiation-specific lipid droplets were virtually excluded from protoporphyrin IX fluorescence. In addition to weak mitochondrial and strong membrane fluorescence, distinctive spots with strong fluorescence were observed. These did not colocalize with fluorescent probes for mitochondria, lysosomes or the Golgi apparati.     

THE EFFECT OF ENVIRONMENTAL LIGHT EXPOSURE ON DRUG-INDUCED PORPHYRIA IN THE RAT

Abstract— The porphyrinogenic drug, 3,5-diethoxycarbonyl 1,4-dihydrocollidine (DDC) when administered orally to rats evoked large increases in hepatic δ-aminolevulinic acid synthetase (ALAS) activity and hepatic protoporphyrin levels. These increases varied markedly according to light exposure patterns of the animals. DDC-treated animals continuously exposed for one week to fluorescent Blacklight lamps (Westinghouse FS-40) demonstrated a greater than two-fold increase in hepatic ALAS and a greater than threefold increase in liver protoporphyrin levels as compared to DDC-treated animals exposed to ambient light-dark cycling. Furthermore, the skin of porphyric animals continuously exposed to light showed larger increases in porphyrin content as compared to rats exposed to ambient light. These studies indicate that light exposure patterns can profoundly alter the activity of the hepatic heme pathway in the rat and suggest that light exposure could play a role in the production of drug-induced porphyria in man.     

Binding studies of porphyrins to human serum albumin using affinity capillary electrophoresis

The present work demonstrates that affinity capillary electrophoresis (ACE) can be employed as a valuable and powerful tool for studying the interactions between porphyrins and proteins in biological and biomedical research, such as the development of porphyrins and related compounds as efficient and selective photosensitizers in the photodynamic therapy of cancers. Binding constants of human serum albumin (HSA) to four biological porphyrins (uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, protoporphyrin IX), which possess a wide range of hydrophobicity, were estimated by ACE. Based on 1:1 molecular association between these individual porphyrins and HSA, the change of the electrophoretic mobility of HSA as a function of porphyrin concentration in the run buffer was measured and the binding constants were calculated from the slope of the Scatchard plots. The binding constant values were found to be 8.80 +/- 0.51 x 10(4) M(-1), 2.39 +/- 0.16 x 10(5) M(-1), 1.61 +/- 0.11 x 10(6) M(-1), and 9.34 +/- 0.30 x 10(6) M(-1) for uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, and protoporphyrin IX, respectively, and most of these results are in good agreement with those reported in the literature using conventional methods for binding measurements. Additionally, experimental binding constant data obtained using ACE was found to exhibit very good correlation with theoretical hydrophobicity values calculated using the Rekker's hydrophobic fragmental constant method, thus further supporting the hypothesis that the hydrophobicity of the porphyrin side chains play an important role in governing the hydrophobic interaction of porphyrins with serum proteins such as HSA.     

THE EFFECT OF ALA AND RADIATION ON PORPHYRIN/HEME BIOSYNTHESIS IN ENDOTHELIAL CELLS

To study porphyrin biosynthesis in human microvascular endothelial cells, HMEC-1 cells, a transformed human microvascular endothelial cell line, were incubated with 5-aminolevulinic acid (ALA), the precursor of endogenous porphyrins, and porphyrin accumulation was measured spectro-fluorometrically. The HMEC-1 cells accumulated porphyrin in a concentration-related and a time-dependent fashion. Protoporphyrin was the predominant porphyrin accumulated in the cells. The effect of light on protoporphyrin accumulation was evaluated by exposing the ALA-loaded HMEC-1 cells to ultraviolet-A (UVA) and blue light, followed by another incubation with ALA for 2–24 h. Enhancement of protoporphyrin accumulation in irradiated HMEC-1 cells was observed 2–24 h after irradiation, which was associated with a decrease in ferrochelatase protein and activity. Porphyrin accumulation from ALA after irradiation was significantly decreased when catalase (750–3000 U/mL, 29.3–44.3% suppression) or superoxide dismutase (270 U/mL, 36.4% suppression) was present during irradiation. These data demonstrate that HMEC-1 cells were capable of porphyrin biosynthesis, and that exposure of protoporphyrin-containing HMEC-1 cells to UVA and blue light, which includes the Soret band spectrum, decreased the ferrochelatase activity and its protein. These changes were mediated, at least in part, by reactive oxygen species.     

Characterization of porphyrins of rat Harderian gland by thin layer chromatography and mass spectrometry: no evidence for a tricarboxylic acid porphyrin

Reversed-phase TLC systems using ion-pairing reagents are described for the separation without prior derivatization of the porphyrins of rat Harderian gland. Porphyrins with R(f) values greater than protoporphyrin but smaller than coproporphyrin, representing harderoporphyrin, were labile to 1 m NaOH, which converted them to protoporphyrin, inconsistent with the established literature that harderoporphyrin is a tricarboxylic acid porphyrin. It was confirmed by HPLC/electrospray ionization MS that this 'harderoporphyrin' fraction consists of the recently characterized glycoconjugate, protoporphyrin-1-O-acyl beta-xyloside, with trace amounts of protoporphyrin-1-O-acyl beta-glucoside.     

Porphyrin profiles in blood, urine and faeces by HPLC/electrospray ionization tandem mass spectrometry

A high-resolution high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry method is described for the analysis of porphyrins in blood, urine and faeces. The gradient elution reversed-phase HPLC system using acetonitrile-methanol-1 m ammonium acetate/acetic acid buffer (pH 5.16) as gradient solvent mixtures was able to separate all porphyrin metabolites, including the type I and type III isomers of uroporphyrin, hepta-, hexa- and penta-carboxylic acid porphyrins and coproporphyrin. The porphyrins were positively identified by the protonated molecules [M+H](+) and further characterized by tandem mass spectrometric analysis with each porphyrin giving a characteristic collisioninduced dissociation product ion spectrum. The mass chromatograms obtained by HPLC/ESI MS are useful for the differential diagnosis of the porphyrias, since each type of porphyria has a typical porphyrin excretion pattern.     

PENETRATION POTENCY OF TOPICAL APPLIED δ-AMINOLEVULINIC ACID FOR PHOTODYNAMIC THERAPY OF BASAL CELL CARCINOMA *

Abstract Penetration potency of δ-aminolevulinic acid (ALA) was studied by examining fluorescence of endogenous protoporphyrin IX in different histological types of basal cell carcinoma. Ten basal cell carcinomas were coated with an ointment containing 10% ALA prior to excision; five served as controls. Tumors were excised either 4 h or 12 h after application of ALA using a modified Mohs’micrographic surgical technique. Horizontal sections were cut from deep dermis to tumor surface and examined under a fluorescence microscope. After 4 h of application, only skin appendages demonstrated fluorescence typical of protoporphyrin IX. After 12 h, fluorescence was detectable in tumor cells in deep dermis. The five controls revealed no fluorescence at any site. These results may confirm the high penetration potential of topically applied ALA and its usefulness in photodynamic therapy. For tumors penetrating to deep dermis, an application time of more than 4 h seems necessary, at least when hydrophilic solvents for ALA are used.     

Photofrin as a specific radiosensitizing agent for tumors: studies in comparison to other porphyrins, in an experimental in vivo model

The use of ionizing radiation for tumor treatment represents a well established therapeutic modality. The efficiency and selectivity of radiotherapeutic protocols can be often enhanced by the addition of specific chemical compounds that optimise the response of the tumor to the incident radiation as compared with peritumoral tissue districts. The results of this study showed that Photofrin, a porphyrin derivative which is presently used as a tumor-photosensitizing agent in photodynamic therapy (PDT), can also act as an efficient tumor radiosensitizer. To test this possibility, we used nude mice subcutaneously implanted with human bladder cancer RT4. The mice were injected with different porphyrin-type photosensitizing agents, including Photofrin, 5-aminolevulinic acid, chlorin e(6), haematoporphyrin, protoporphyrin, Zn-tetrasulphophtalocyanine, and irradiated with 5 and 15 Gy using a Siemens X-ray device. Even though all the porphyrins accumulated in significant amounts in the neoplastic lesion, only Photofrin significantly improved the response of the tumor to irradiation by increasing the doubling time of the tumor volume from 6.2 days in the untreated control group to 10.9 days in the 5 and 15 Gy-irradiated groups. The tumor response was maximal with injected Photofrin doses of 7.5 mg/kg, and was not further enhanced by injection of higher doses. Our hypothesis is, that the radiosensitizing effect of Photofrin seems to be due to some oligomeric constituents which could specifically react with radiogenerated-radicals thereby amplifying the effect of the X-ray radiation.     

PHOTOSENSITIZATION OF MURINE TUMOR, VASCULATURE and SKIN BY 5-AMINOLEVULINIC ACID-INDUCED PORPHYRIN

Abstract— The effects of topical and systemic administration of 5-aminolevulinic acid (ALA) were examined in several murine tumor systems with regard to porphyrin accumulation kinetics in tumor, skin and blood, vascular and tumor cell photosensitization and tumor response after light exposure. Marked, transient increases in porphyrin levels were observed in tumor and skin after systemic and topical ALA. Rapid, transient, dose-dependent porphyrin increases were also observed in blood; these were pronounced after systemic ALA injection and mild after topical application. They were highest within 1 h after ALA injection, thereafter declining rapidly. This matched the clearing kinetics of injected exogenous protoporphyrin IX (PpIX). Initially, vascular photosensitivity changed inversely to blood porphyrin levels, increasing gradually up to 5 h post-ALA, as porphyrin was clearing from the bloodstream. This pattern was again matched by injected, exogenous PpIX. After therapeutic tumor treatment vascular disruption of the tumor bed, while observed, was incomplete, especially at the tumor base. Minimal direct tumor cell kill was found at low photodynamic therapy (PDT) doses (250 mg/kg ALA, 135 J/cm² light). Significant, but limited (<1 log) direct photodynamic tumor cell kill was obtained when the PDT dose was raised to 500 mg/kg systemic ALA, followed 3 h later by 270 J/cm², a dose that was however toxic to the animals. The further reduction of clonogenic tumor cells over 24 h following treatment was moderate and probably limited by the incomplete disruption of the vasculature. Tumor responses were highest when light treatment was carried out at the time of highest tumor porphyrin content rather than at the time of highest vascular photosensitivity. Tumor destruction did not reach the tumor base, regardless of treatment conditions.     

Assessing the In Vitro Activity of Selected Porphyrins in Human Colorectal Cancer Cells

Standard in vitro analyses determining the activity of different compounds included in the chemotherapy of colon cancer are currently insufficient. New ideas, such as photodynamic therapy (PDT), may bring tangible benefits. The aim of this study was to show that the biological activity of selected free-base and manganese (III) metallated porphyrins differs in the limitation of colon cancer cell growth in vitro. White light irradiation was also hypothesized to initiate a photodynamic effect on tested porphyrins. Manganese porphyrin (>1 μM) significantly decreased the viability of the colon tumor and normal colon epithelial cells, both in light/lack of light conditions, while decreasing a free-base porphyrin after only 3 min of white light irradiation. Both porphyrins interacted with cytostatics in an antagonistic manner. The manganese porphyrin mainly induced apoptosis and necrosis in the tumor, and apoptosis in the normal cells, regardless of light exposure conditions. The free-base porphyrin conducted mainly apoptosis and autophagy. Normal and tumor cells released low levels of IL-1β and IL-10. Tumor cells released a low level of IL-6. Light conditions and porphyrins were influenced at the cytokine level. Tested manganese (III) metallated and free-base porphyrins differ in their activity against human colon cancer cells. The first showed no photodynamic, but a toxic activity, whereas the second expressed high photodynamic action. White light use may induce a photodynamic effect associated with porphyrins.     

SENSITIVITY OF HemA MUTANT Escherichia coli CELLS TO INACTIVATION BY NEAR-UV LIGHT DEPENDS ON THE LEVEL OF SUPPLEMENTATION WITH δ-AMINOLEVULINIC ACID

Abstract— Four strains carrying all four possible combinations of the alleles nur, nur⁺, uvr A6 and uvr A ⁺ were transduced to hemA8 . The hemA8 mutation blocks the synthesis of δ-aminolevulinic acid (δ-ALA), one of the first steps in the synthesis of porphyrin and, ultimately, cytochromes essential for aerobic respiration. The cells were grown either with or without δ-ALA and treated with broad-spectrum near-ultraviolet light (NUV; 300–400 nm). hemA8 defective cells grown without δ-ALA were resistant to inactivation by NUV while hemA8 cells were sensitive to such inactivation when supplemented with δ-ALA. The sensitivity to NUV inactivation conferred by the nur gene was retained in the hemA8 derivatives. The sensitivity of such cells to NUV inactivation can be controlled by varying the level of δ-ALA supplementation. The level of δ-ALA supplementation did not influence the sensitivity of the cells to inactivation by far-UV light (FUV; 200–300 nm). The near-UV sensitivity of hemA⁺ cells was not significantly altered when grown with δ-ALA suppiementation suggesting that endogenously formed δ-ALA supports the normal, regulated level of porphyrin synthesis. These results can be interpreted to mean that porphyrin components of the respiratory chain in E. coli represent chromophores involved specifically in broad-spectrum NUV inactivating events.     

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.     

The application of a compact multispectral imaging system with integrated excitation source to in vivo monitoring of fluorescence during topical photodynamic therapy of superficial skin cancers

A novel, compact and low-cost multispectral fluorescence imaging system with an integrated excitation light source is described. Data are presented demonstrating the application of this method to in vivo monitoring of fluorescence before, during and after topical 5-aminolevulinic acid photodynamic therapy of superficial skin cancers. The excitation source comprised a fluorescent tube with the phosphor selected to emit broadband violet light centered at 394 nm. The camera system simultaneously captured spectrally specific images of the fluorescence of the photosensitizer, protoporphyrin IX, the illumination profile and the skin autofluorescence. Real-time processing enabled images to be manipulated to create a composite image of high contrast. The application and validation of this method will allow further detailed studies of the characteristics and time-course of protoporphyrin IX fluorescence, during topical photodynamic therapy in human skin in vivo.