Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.     

In vivo mTHPC photobleaching in normal rat skin exhibits unique irradiance-dependent features

We report measurements performed on the normal skin of rats in vivo, which provide information on the photobleaching kinetics and mechanisms of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC). Loss of mTHPC fluorescence was monitored using in vivo fluorescence spectroscopy during photodynamic therapy (PDT) performed using 650 nm laser irradiation. The bleaching was evaluated for irradiances of 5, 20 and 50 mW cm(-2). Two distinct phases of mTHPC photobleaching were observed. In the first phase there was no obvious irradiance dependence in the loss of fluorescence vs fluence. The second phase was initiated by an irradiance-dependent discontinuity in the slope of the bleaching curve, after which the photobleaching rates showed an irradiance dependence consistent with an oxygen-dependent reaction process. To investigate the unusual shape of the in vivo bleaching curves, we measured the PDT-induced changes in O2 concentrations in mTHPC-sensitized spheroids irradiated with 2, 5 and 20 mW cm(-2) of 650 nm light. The oxygen concentration data indicated no unusual features within the range of fluences where the discontinuities in fluorescence were observed during in vivo spectroscopy. The fluorescence from the in vivo bleaching experiments thus reports a phenomenon that is not reported by measurements of the photochemical oxygen consumption in the spheroids.     

Monitoring ALA-induced PpIX photodynamic therapy in the rat esophagus using fluorescence and reflectance spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg(-1) was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm(-2) using a total fluence of 54 J cm(-2). Forty-eight hours after PDT the esophagus was excised and histologically examined for PDT-induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two-phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two-phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.     

Protoporphyrin IX fluorescence photobleaching and the response of rat Barrett's esophagus following 5-aminolevulinic acid photodynamic therapy

Barrett's esophagus (BE) can experimentally be treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT), in which ALA, the precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) and subsequent irradiation with laser light are applied to destroy the (pre)malignant tissue. Accurate dosimetry is critical for successful ALA-PDT. Here, in vivo dosimetry and kinetics of PpIX fluorescence photobleaching were studied in a rat model of BE. The fluence and fluence rate were standardized in vivo and PpIX fluorescence was measured simultaneously at the esophageal wall during ALA-PDT and plotted against the delivered fluence rather than time. Rats with BE were administered 200 mg kg(-1) ALA (n = 17) or served as control (n = 4). Animals were irradiated with 633 nm laser light at a measured fluence rate of 75 mW cm(-2) and a fluence of 54 J cm(-2). Large differences were observed in the kinetics of PpIX fluorescence photobleaching in different animals. High PpIX fluorescence photobleaching rates corresponded with tissue ablation, whereas low rates corresponded with no damage to the epithelium. Attempts to influence tissue oxygenation by varying balloon pressure and ventilation were shown not to be directly responsible for the differences in effect. In conclusion, in vivo dosimetry is feasible in heterogeneous conditions such as BE, and PpIX fluorescence photobleaching is useful to predict the tissue response to ALA-PDT.     

Monitoring in situ dosimetry and protoporphyrin IX fluorescence photobleaching in the normal rat esophagus during 5-aminolevulinic acid photodynamic therapy

Experimental therapies for Barrett's esophagus, such as 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), aim to ablate the premalignant Barrett's epithelium. However, the reproducibility of the effects should be improved to optimize treatment. Accurate irradiation with light of a proper wavelength (633 nm), fluence and fluence rate has shown to be critical for successful ALA-PDT. Here, we have used in situ light dosimetry to adjust the fluence rate measured within the esophagus for individual animals and monitored protoporphyrin IX (PpIX) fluorescence photobleaching simultaneously. Rats were administered 200 mg kg-1 ALA (n = 14) or served as control (n = 7). Animals were irradiated with an in situ measured fluence rate of 75 mW cm-2 and a fluence of 54 J cm-2. However, this more accurate method of light dosimetry did not decrease the variation in tissue response. Large differences were also observed in the dynamics of PpIX fluorescence photobleaching in animals that received the same measured illumination parameters. We found that higher PpIX fluorescence photobleaching rates corresponded with more epithelial damage, whereas lower rates corresponded with no response. A two-phased decay in PpIX fluorescence could be identified in the response group, with a rapid initial phase followed by a slower rate of photobleaching. Non-responders did not show the rapid initial decay and had a significantly lower rate of photobleaching during the second phase of the decay (P = 0.012).     

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.     

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/cm² 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.     

Monitoring PDT by means of superficial reflectance spectroscopy

Monitoring of relevant parameters during photodynamic therapy (PDT) and correlating these with treatment response is necessary to guarantee optimal and reproducible treatment outcome. In this paper we study the correlation between changes in the local tissue optical properties (absorption and scattering coefficients) during ALA-PDT and changes in PpIX fluorescence. The optical properties are measured extremely superficially by employing a single fiber for the delivery and collection of white light to and from the tissue. The measured reflectance spectrum is modeled in terms of four relevant parameters: blood saturation, relative blood volume fraction, scattering intensity and wavelength dependence of the scattering. All these parameters, except the relative blood volume fraction, are shown to correlate with the rate of photobleaching of PpIX, which in turn has previously been shown to correlate with the response of tissues to PDT. These results yield valuable insight in the behavior of these parameters during PDT and their suitability to predict PDT-response for other photosensitizers for which monitoring through photobleaching is not possible.     

Photobleaching of hypericin bound to human serum albumin,cultured adenocarcinoma cells and nude mice skin

Hypericin is a promising photosensitizer for photodynamic therapy (PDT) characterized by a high yield of singlet oxygen. Photobleaching of hypericin has been studied by means of absorption and fluorescence spectroscopy in different biological systems: in human serum albumin solution, in cultured human adenocarcinoma WiDr cells and in the skin of nude mice. Prolonged exposure to light (up to 95 min, 100 mW/cm2) of wavelength around 596 nm induced fluence-dependent photobleaching of hypericin in all studied systems. The photobleaching was not oxygen dependent, and singlet oxygen probably played no significant role. Emission bands in the spectral regions 420-560 nm and above 600 nm characterize the photoproducts formed. An emission band at 615-635 nm was observed after irradiation of cells incubated with hypericin or of mouse skin in vivo but not in albumin solution. The excitation spectrum of these products resembled that of hypericin. Hypericin appears to be more photostable than most sensitizers used in PDT, including mTHPC and Photofrin.     

Comparison of the photodynamic effect of exogenous photoprotoporphyrin and protoporphyrin IX on PAM 212 murine keratinocytes

A comparative study of the cellular photosensitizing properties of protoporphyrin IX (PpIX) and photoprotoporphyrin (Ppp) was carried out in the transformed murine keratinocyte cell line, PAM 212. Time-course fluorescence studies were performed to determine the rate of uptake by cells together with fluorescence microscopy. The sensitized cells were laser irradiated with a range of light doses at 635 or 670 nm to determine the phototoxicity of the two compounds and to investigate their relative fluorescence photobleaching properties. Ppp showed enhanced phototoxicity at both its optimal activation wavelength of 670 nm (eight times more phototoxic than PpIX activated at its optimal wavelength of 635 nm for the same fluence) and at 635 nm (three times more phototoxic than PpIX at the same wavelength), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The photobleaching rate of Ppp in cells was found to be higher using 670 nm irradiation compared with that of PpIX at 635 nm irradiation. At 635 nm, however, the photobleaching rate of Ppp was comparable to that of PpIX. The photobleaching quantum yields of the two compounds in cells were found to be similar at approximately 5 x 10(-4), with the same value confirmed at both 670 and 635 nm irradiation for Ppp. The fluorescence lifetime of Ppp in cells was measured as 5.4 ns using time-correlated single photon counting.     

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.     

Fluence Rate-Dependent Photobleaching of Intratumorally Administered Pc 4 Does not Predict Tumor Growth Delay

We examined effects of fluence rate on the photobleaching of the photosensitizer Pc 4 during photodynamic therapy (PDT) and the relationship between photobleaching and tumor response to PDT. BALB/c mice with intradermal EMT6 tumors were given 0.03 mg kg^?1 Pc 4 by intratumor injection and irradiated at 667 nm with an irradiance of 50 or 150 mW cm^?2 to a fluence of 100 J cm^?2. While no cures were attained, significant tumor growth delay was demonstrated at both irradiances compared with drug‐only controls. There was no significant difference in tumor responses to these two irradiances (P = 0.857). Fluorescence spectroscopy was used to monitor the bleaching of Pc 4 during irradiation, with more rapid bleaching with respect to fluence shown at the higher irradiance. No significant correlation was found between fluorescence photobleaching and tumor regrowth for the data interpreted as a whole. Within each treatment group, weak associations between photobleaching and outcome were observed. In the 50 mW cm^?2 group, enhanced photobleaching was associated with prolonged growth delay (P = 0.188), while at 150 mW cm^?2 this trend was reversed (P = 0.308). Thus, it appears that Pc 4 photobleaching is not a strong predictor of individual tumor response to Pc 4‐PDT under these treatment conditions.     

Fractionated Illumination at Low Fluence Rate Photodynamic Therapy in Mice

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

Photobleaching of Arterial Fluorescent Compounds: Characterization of Elastin, Collagen and Cholesterol Time-resolved Spectra during Prolonged Ultraviolet Irradiation

To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72, 7.25 and 21.75 microW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence photobleached the most, and elastin fluorescence photobleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly distorted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy.     

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.     

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.     

Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.     

Characterization of Photodegradation of Meta-tetra (Hydroxyphenyl)chlorin (mTHPC) in Solution: Biological Consequences in Human Tumor Cells

The photobleaching of meta -tetra(hydroxyphenyl)chlorin m THPC) (irradiation wavelength 413 nm) in protein-containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (λ_exc= 423 nm, λ_em= 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325–450 nm. The rate of m THPC photodegradation, followed by decay in absorbance, was 15-fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of m THPC. In order to determine the consequences of photobleaching of fluorescing m THPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached m THPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of m -THPC-photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated m -THPC plays an important role in m THPC-PDT.     

Correlation between oxygen consumption and photobleaching during in vitro photodynamic treatment with ATX-S10.Na(II) using pulsed light excitation: dependence of pulse repetition rate and irradiation time

We revealed that in ATX-S10.Na(II)(13,17-bis (1-carboxypropionyl) carbamoylethyl-8-etheny-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetraethyl porphyrin sodium)-mediated photodynamic therapy using 667 nm nanosecond-pulsed light excitation at a peak intensity of 2.0 MW/cm(2), phototoxicity increased with decreasing pulse repetition rate in the range of 5-30 Hz for A549 cell cultures. To examine the relation between the reaction mechanism and measured phototoxicity, we carefully measured the kinetics of photochemical oxygen consumption and photobleaching during irradiation of ATX-S10.Na(II)-sensitized A549 monolayer cultures. Measurements of oxygen consumption with a microelectrode, which was performed just above the cells, showed that there was no significant difference between the magnitudes of decrease in oxygen at the three repetition rates at the same cumulative fluence. Loss of ATX-S10.Na(II) fluorescence intensity also exhibited little repetition rate dependence when compared at the same cumulative fluence. We investigated the correlation between oxygen consumption and photobleaching during irradiation and obtained "fluorescence-oxygen diagrams." The diagrams showed dynamic changes between oxygen-dependent and oxygen-independent photobleaching at the higher repetition rates of 10 and 30 Hz, whereas such change was not clearly seen over the whole irradiation time at 5 Hz. These results suggest that the reduced phototoxicity at high repetition rates might be due to an oxygen-independent reaction. We presumed that the change in the reaction mechanism was associated with the local concentrations of the photosensitizer and oxygen in cells during irradiation.     

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.