LiWei Ma

Topical application of 5‐aminolaevulinic acid, methyl 5‐aminolaevulinate and hexyl 5‐aminolaevulinate on normal human skin

Backgroundu2002 5‐Aminolaevulinic acid (ALA) and its ester derivatives are used in photodynamic therapy. Despite extensive investigations, the differences in biodistribution and pharmacokinetics of protoporphyrin IX (PpIX) induced by ALA and its derivatives are still not well understood, notably for humans.

The photosensitizing effect of the photoproduct of protoporphyrin IX.

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm(2) light at 635 nm, the photoproduct--the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.

The Temperature Dependence of Protoporphyrin IX Production in Cells and Tissues

The formation of protoporphyrin IX (PpIX) in human skin during topical application of 5‐aminolevulinic acid (ALA) was found to be strongly temperature dependent, with an activation energy of about 17 kcal/mol. This temperature dependence is mainly related to porphyrin production and not to ALA penetration into the skin. The penetration of ALA into mouse and human skin was almost temperature independent. The activation energy of PpIX production in mouse skin was practically identical with that in human skin. The activation energy of ALA uptake by cells in vitro was about 10 kcal/mol and that for PpIX production was about 13 kcal/mol. The latter activation energy was within the error limits similar to that for the activity of the enzyme porphobilinogen deaminase, suggesting that this enzyme might represent a rate‐limiting step for PpIX production in living tissue.

Combined Treatment of Ionizing Radiation and Photosensitization by 5-Aminolevulinic Acid-Induced Protoporphyrin IX

The response of human colon adenocarcinoma cells of the line WiDr to the combined treatment of ionizing radiation and photosensitization by 5-aminolevulinic acid-induced protoporphyrin IX was assessed by a colony-forming assay. A dose of X rays inactivating approximately 50% of the cells was used. Seventy to 85% of the cells accumulated in S and G2 + M phase 12-24 h after such a treatment as measured by flow cytometry, while the distribution of cells in the phases of the cell cycle approached that of untreated cells 48 h after X-ray treatment. Cellular photosensitization was developed by endogenous synthesis of protoporphyrin IX (PPIX) from the precursor 5-aminolevulinic acid (5-ALA). This was performed by treating the cells with 1 mM 5-ALA for 4 h in a serum-free medium. The endogenous synthesis of PPIX increased with time after the cells had been subcultured, i.e. the ability of the cells to synthesize PPIX increased 1.5-2-fold within 48 h of incubation. This was not due to effects of trypsin on the cells. Photochemotherapy with 5-ALA was given 0-48 h after X rays. The combined cytotoxic effect was analyzed by an isobologram after correction of the survival curves for microcolony formation and differences in intracellular concentration of PPIX. The results indicate that 5-ALA PCT given 0-4 h after X rays acts slightly antagonistically while 5-ALA PCT given 12-48 h after X rays acts slightly synergistically.

Choice of the proper wavelength for photochemotherapy

All photosensitizers applied in experimental- and clinical-photochemotherapy (PCT) have broad absorption spectra stretching from the ultraviolet up to 6 - 700 nm. Light of wavelengths in the red part of the spectrum is chosen for PCT even though the extinction coefficients of the sensitizers are usually smaller in this wavelength region than at shorter wavelengths. Thus, if one wants to treat superficial tumors or skin disorders, this may be a wrong choice. Two pieces of information are needed in order to make a proper choice of wavelength to treat a lesion of a given depth: the wavelength dependence of the optical penetration depth into tissue, and the action spectrum for tumor destruction. Additionally, the skin photosensitivity induced by the drug should be considered. We have non-invasively measured the optical penetration spectra of human tissues in vivo and the fluorescence excitation spectra for several sensitizers, including protoporphyrin (PpIX), in cells. Assuming that the action spectrum for cell inactivation can be approximated by the fluorescence excitation spectrum of the sensitizer -- which is indeed the case for a number of sensitizers in cells in vitro -- we have considered the situation for 5-aminolevulinic acid-induced PpIX in human tissue. All the way down to about 2 mm below the surface light in the Soret band (-410 nm) would give the largest cell inactivation, while at depth exceeding 2 mm, the conventional 635 nm light would be optimal. Light at the argon laser wavelength 514.5 nm is more efficient than light at 635 nm down to 1 mm. From the surface and down to 6 mm, the 635 nm peak of the excitation spectrum of PpIX, as evaluated per photon incident on the skin surface, is redshifted by less than 2 nm. In some cases photosensitizing photoproducts are formed during PCT, such as photoprotoporphyrin during PCT with PpIX. In such cases it may be advantageous to apply a broad-band light source with a spectrum that covers also part of the action spectrum of this photoproduct.

Production of protoporphyrin IX induced by 5-aminolevulinic acid in transplanted human colon adenocarcinoma of nude mice can be increased by ultrasound.

BALB/c nude mice bearing WiDr human colon adenocarcinoma were used to determine the effect of ultrasound on the production of 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) both in the tumors and in skin overlying the tumors. Ultrasound (1 MHz) with pulsed irradiation at an average intensity of 3 W/cm2 was given 10 min to the tumor area 10 min after administration of ALA (20% in an oil‐in‐water emulsion applied topically on the surface of the tumor for 30 min to 3 hr). An approximately 45% increase in the amount of PpIX produced by ALA in the tumors was obtained within 1 to 2 hr following ultrasound treatment. In particular, 1 hr after ultrasound treatment, the amount of PpIX in the tumors was at the same level as that 3 hr after ALA application alone. However, pulsed ultrasound irradiation for 5 min or continuous irradiation for 5 or 10 min had no significant effect on the production of PpIX by the tumor 1 hr after topical ALA application. Furthermore, in most cases, the amount of PpIX in the tumors was significantly decreased when ultrasound was given immediately before ALA application. There was no significant change in the ratio of the amount of PpIX in tumor to that in skin after ultrasound treatment. Most likely, the distribution of PpIX fluorescence in the tumors treated with ultrasound was more homogeneous than that in the tumors given ALA only. Our results provide a theoretical basis for possible clinical use of ultrasound‐combined ALA or ALA based photodynamic therapy. Int. J. Cancer 78:464–469, 1998.

Potentiation of Photodynamic Therapy by Mitomycin C in Cultured Human Colon Adenocarcinoma Cells

The effects of photodynamic therapy (PDT) alone and in combination with Mitomycin C (MMC) on WiDr cells, a human colon adenocarcinoma cell line, were investigated. The addition of MMC increased the cytotoxicity of PDT. The presence of MMC resulted in a reduction or a removal of the shoulder of the PDT survival curves as well as an increase in their slopes. Increasing with the concentrations of MMC from 0.01 to 0.025 micrograms/ml, the cytotoxic effects of the two treatments changed from additivity to supra-additivity as judged by comparing dose-response curves for each treatment alone with survival curves after combination therapy and by isobologram analysis. The cytotoxicity of MMC could also be enhanced by a practically nontoxic treatment of PDT (8% cell inactivation). The cytotoxicities of MMC and PDT in combination were found to be dependent on the sequence of the two treatments. When MMC (> or = 0.02 micrograms/ml) and Photofrin II were given simultaneously for 16 h and then followed by irradiation, the combination was found to be more effective than when MMC was given to the cells immediately after PDT and kept in the medium for 16 h. Possible mechanisms of the combination effects of PDT and MMC are discussed briefly.

In vivo fluorescence of phthalocyanines during light exposure.

Nude mice were given AlPcS2a (aluminum phthalocyanine disulfonate) and AlPcS4 (aluminum phthalocyanine tetrasulfonate) by intraperitoneal injections. After time intervals of 1-48 hours the mice were exposed to 150 mW cm-2 light at 670 nm and the phthalocyanine fluorescence was measured during light exposure. During the first few minutes of light exposure the phthalocyanine fluorescence of the skin of the mice increased by up to a factor of two, indicating lysosomal localization of the dye and permeabilization of the lysosomes. The process did not occur in the skin of dead mice, indicating that the process was dependent on oxygen.

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).

Influenza, solar radiation and vitamin D

The annual death numbers of influenza and pneumonia in Norway were studied for the time period 1980 - 2000. No direct relationships were found with the variations of the annual UVB fluences, probably due to the fact that these variations did not exceed 30 %. However, there was a very pronounced seasonal variation of both influenza deaths and pneumonia deaths, the vast majority occurring during the winter. Vitamin D levels were also estimated from several publications. The data support the hypothesis that a high vitamin D level, as that found in the summer, acts in a protective manner with respect to influenza as well as pneumonia. The findings are discussed and compared with data from tropical and subtropical areas.