Search

Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.

The kinetics of protoporphyrin IX (PpIX) production in normal tissues and WiDr tumors of mice were studied after topical application of 5-aminolevulinic acid (ALA) and its methyl ester and hexyl ester. ALA and ALA esters were applied on a spot of 1.0 cm diameter on normal skin and on skin overlaying tumors. PpIX production was studied by fluorescence measurements. ALA induced PpIX not only on the spot of application but also on remote skin areas. This was not found for the ALA esters. They produced PpIX only on the spot of application. Thus, ALA, but neither its esters nor PpIX, is passing into the circulation. The time needed for ALA to enter the circulation through normal skin was about 5 hr. Even when looking normal, the skin overlaying tumors was more permeable to ALA than normal skin. Thus, when applied on the tumor, ALA induced PpIX on remote skin areas without any lag phase. Mainly, PpIX was found in all tissues although small amounts of a porphyrin with an excitation peak at about 400 nm, supposedly uroporphyrin and/or coproporphyrin, were found, notably in remote skin areas. An altered stratum corneum of the skin overlaying tumors probably contributes to the tumorselectivity, although in the present tumor system less PpIX was found in tumors than in muscles. This is probably related to biochemical and physiological conditions in this particular tumor, since i.p. injection of ALA also leads to less PpIX formation in the tumor than in skin/muscle tissue. Nevertheless, it seems evident that ALA can diffuse more easily from the skin surface and down to the vasculature in the tumor than in the normal tissue and that this leads to a higher concentration of PpIX in the tumor than would have been found if the physiological factors relevant for drug diffusion were the same for tumors as for skin/muscles. © 2001 Wiley-Liss, Inc.

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.

Normal skin of nude mice (Balb/c) was treated topically with 5-aminolevulinic acid (ALA) and its methyl ester (ALA-Me) for 24 hours. Approximately 0.1 gram of freshly prepared cream was applied to a spot of 1 cm2 on the flank of the mice, which was then covered with a transparent dressing. The ALA induced protoporphyrin IX (PpIX) was studied by means of a noninvasive fiber-optic fluorescence probe connected to a luminescence spectrometer. The excitation wavelength was 407 nm, and the emission wavelength was 637 nm. For the first hour a slight lag in PpIX production was observed for the mice treated with ALA-Me compared to the mice treated with ALA. After approximately 12 hours the ALA and the ALA-Me treated mice showed the same PpIX fluorescence intensity. From 12 hours until 24 hours the PpIX fluorescence intensity decreased for both treatment modalities, even though ALA and ALA-Me were continuously present. At 24 hours ALA-Me-treated mice had less than half the amount of PpIX in their skin compared with ALA- treated mice.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The clearance of protoporphyrin IX (PpIX) from the skin of hairless BALB/c mice after topical application of 5- aminolevulinic acid (ALA) and its methyl ester (ALA-Me) was investigated. Creams containing 2 or 20% of ALA or ALA-Me were topically applied on spots of approximately 1 cm2 for 12 hours. The PpIX fluorescence was detected by the means of a Perkin Elmer LS50B luminescence spectrometer equipped with a fiber-optic probe. The emission spectrum was identical with that of cell-bound PpIX. After 12 hours application of ALA and ALA-Me similar amounts of PpIX were found. After creme removal the ALA-induced PpIX fluorescence decayed with a half-life of about 20 hours (20% ALA cream). The ALA-Me-induced PpIX was faster cleared from the skin than ALA-induced PpIX, and had a half-life of about 7 hours (20% ALA-Me cream).© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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. © 1998 Wiley-Liss, Inc.

Nude mice were given AlPcS 2a (aluminum phthalocyanine disulfonate) and AlPcS 4 (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.

Using BALB/c nude mice bearing WiDr human colon adenocarcinoma, we investigated photobleaching and photochemotherapeutic (PCT) effects of meso- tetrahydroxyphenyl-chlorin (mTHPC) and meso- tetrahydroxyphenyl-bacteroiochlorin (mTHPBC). For both studies, mice were injected i.p. 1 mg/kg mTHPC and mTHPBC, respectively, 24 hr before light irradiation. Photobleaching of the sensitizers in mouse skin was carried out using a dye laser (for mTHPC) and 1 KW xenon source equipped with a monochromator (for mTHPBC). Fluorescence measurements were made by means of a fiberoptic system, exciting and collecting the fluorescence from the mouse skin. The system was coupled to a PE L550 fluorimeter. For the PCT study, the mTHPC or mTHPBC-sensitized tumors were exposed to the laser at a fluence of 10 J/cm2. The responses of the treated tumors were evaluated by measuring the tumor growth. We found that both mTHPC and mTHPBC are photolabile. Approximately 80% of the fluorescence of the two dyes in the mouse skin is bleached by a fluence of 10 J/cm2. When mTHPBC in mouse skin was photobleached by light of 740 nm, the bacteriochlorin (peak at 740 nm) was significantly bleached while the chlorin (peak at 652 nm) was unaffected. The growth of the tumors was delayed by 13 days after PCT with 1 mg/kg mTHPC Irradiation at 652 nm) and 5 days delay after PCT with 1 mg/kg mTHPBC (irradiation at 515 nm). The present data indicate that (1) by proper choice of a low dose of mTHPC or MTHPBC, it is possible to photobleach the sensitizers in normal tissues without eliminating their PCT effect on tumor tissues; (2) in order to increase the efficiency of PCT with mTHPBC, fractionated irradiation with different wavelengths (740 nm and 652 nm) should be considered to be used.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A main factor to consider before photochemotherapy (PCT) is chosen to treat a tumor is whether sufficient fluences of light can reach the base of the tumor. The choice of optimal wavelengths for PCT should also be based on a knowledge of the optical penetration spectrum of the tumor. By means of a fiberoptic probe, adapted to a commercial fluorescence spectrometer, it is possible to measure the penetration depths of visible light into living, human tissues. We have performed such experiments, using the light from a 1000 W Xenon lamp. The light was guided from the lamp to the tissue by a perspex rod and the fluence rate penetrating different thickness of tissue was monitored by the fiberoptic probe. From penetration curves obtained at a number of wavelengths, it was possible to construct an accurate optical penetration spectrum of the tissue.

Combination effects of photodynamic therapy (PDT) with meso-tetra (di-adjacent-sulfonatophenyl)porphine (TPPS2a) and the microtubule (MT) inhibitor, vincristine (VCR), were studied in the CaD2mouse tumor model in mice. A synergistic effect was found when VCR, at an almost nontoxic dose (1mg/kg), was injected i.p. into the mice 6 hr before PDT. The thta on mitotic index show a 4-5 fold accumulation of the cells in mitosis 6 hr after injection of VCR into the mice. Cell cycle and ploidydistributions in tumor tissues were determined by means of image analysis with measurement of integrated optical density after Feulgen reaction on monolayers. Ploidy distribution of the tumors was not significantly changed 6 and 12 hr after administration of VCR only, while an increasing aneuploidy was observed 24 and 48 hr after VCR treatment. No prominent changes of the cell cycle and ploidy distributions were found in the tumor tissues after PDT or PDT combined with VCR. Keywords: Photodynamic therapy, Microtubule, Sulfonated tetraphenyl porphines, Vincristine.2. INTRODUCTIONIt has been demonstrated that PDT with a number of dyes [Photofrmn, tetra (3-hydroxyphenyl)

Some key data concerning the pharmacokinetics of PCT photosensitizers are reviewed. The following topics are discussed: The binding of photosensitizers to serum proteins, and the significance of LDL binding for tumor localization, the distribution of sensitizers among different tissue compartments and the significance of extracellular proteins and other stromal elements, such as macrophages, low tumor pH, leaky vasculature and poor lymphatic drainage for tumor selectivity of drugs, the retention and excretion of sensitizers, and intracellular pharmacokinetics. Furthermore, the usefulness of fluorescence measurements in the study of sensitizer pharmacokinetics is briefly discussed. A key observation is that 1O2 has a short radius of action. Since practically all PCT sensitizers act via the 1O2 pathway, only targets with significant sensitizer concentrations can be damaged. A given number of 1O2 entities generated in different organelles (mitochondria, lysosomes, plasma membrane, etc.) may lead to widely different effects with respect to cell inactivation. Similarly, sensitizers localizing in different compartments of tissues may have different photosensitizing efficiencies even under conditions of a similar 1O2 yield.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

All photosensitizers applied in experimental and clinical photochemotherapy (PCT) of cancer are degraded during light exposure. Under certain conditions this may be a disadvantage since larger light fluences are needed to destroy the malignant tissue. However, photodegradation may also offer an advantage: if the applied dose of sensitizer is so low that most of it is photodegraded before normal tissue is destroyed, but still large enough to sensitize the tumor to destruction, one may achieve a larger tumor to normal tissue therapeutic ratio than when using a higher dose of sensitizer. Tumors usually contain two to ten times higher concentrations of sensitizers than do the surrounding normal tissues. We have studied the photodegradation of a number of sensitizers, including Photofrin (PII), benzoporphyrin derivative mono acid ring A (BPD), chlorin e6 (Chle6) 5-aminolevulinic acid (ALA)- induced protoporphyrin IX (PpIX), meso-tetrahydroxyphenyl-chlorin (m-THPC), meso- tetrahydroxyphenyl-porphyrin (m-THPP) tetraphenylporphine tetrasulfonated (TPPS4), aluminum phthalocyanine disulfonated (AlPcS2), tetrasulfonated (AlPcS4) and zinc phthalocyanine (ZnPc) in liposomes. The sensitizers were injected in Balb/c nude mice and exposed to light from an argon pumped dye laser, tuned to the appropriate therapeutic wavelength at a fluence rate of 100 mW/cm2. The sensitizer fluorescence in the laser- exposed skin was monitored by a fiberoptic probe coupled to a fluorescence spectrometer. The kinetics of the fluorescence decay during PCT were, in all cases, nonexponential but differed from dye to dye. Chle6 and m-THPC were found to be the most photolabile sensitizers. AlPcS4 and AlPcS2 and, to a minor degree, TPPS4 showed a peculiar fluorescence increase during PCT, similar to what we have found earlier for these sensitizers in cells in vitro. The fluorescence increase is indicative of lysosomal localization and perforation of the lysosomes during PCT.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Topically applied ALA-PDT for human nodular basal cell carcinoma has encountered some problems, one of which is that the concentration of ALA-induced PpIX is in some cases too low to sensitize complete tumor destruction. Our present study, by means of an optical-fiber based point monitoring system in situ, has shown that the fluorescence intensity of PpIX was significantly higher in the normal skin of nude mice after topical application of ALA and desferrioxamine (DF) or in combination with DMSO than that of ALA alone or ALA plus DMSO. These data indicate that DF has an effect on enhancing the production of ALA- induced PpIX in the mouse skin. Thus, there may clinically be advantages of using ALA and DF over ALA alone in PDT of superficial skin lesions.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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 kinetics of accumulation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its methylester (5-aminolevulinic acid methylester [ALA-Me]) was studied on rat oral mucosa. The accumulation of PpIX in mucosa and skin after intravenous injection of ALA and ALA-Me was also studied. The elimination rate of PpIX was dependent on drug and dose as well as on administration route. Application of ALA on rat oral mucosa and skin caused a systemic effect with PpIX building up in remote skin sites not exposed to the drugs. No such systemic effect was seen after application of ALA-Me either in mucosa or on skin. Intravenous injection of the drugs (0.2 g/kg) leads to more fluorescence in the skin than topical application of the drug (20%). For mucosa, the opposite is true. Maximal PpIX fluorescence appeared later after application of high concentrations of the drugs (around 8 h for 5% and 20% wt/wt) than after application of low concentrations (around 3-5 h for 1% and 2% wt/wt).

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.

The rates of protoporphyrin IX (PpIX) photodegradation and reappearance after light exposure at 420 and 632 nm were measured in mouse skin at different times after 1 h topical application of 5-aminolevulinic acid methyl ester (ALA-Me). After ALA-Me application (1 h) and removal, the fluorescence of PpIX increased for about 1 h, and then reached a maximum and started to decrease. Reappearance of PpIX fluorescence after exposures (degrading 60%-80% of the PpIX) was faster for exposures 0.5 h after ALA-Me application than for exposures 3 h. The bleaching rate was largest in the former case. This indicates that PpIX is located deeper in the skin after 3 h than after 0.5 h, whereas the pool of ALA-Me in the skin is largest at 0.5 h. In all cases, the reappearance was faster at a skin temperature of 35 degrees C than at 23 degrees C. Reappearance of PpIX fluorescence was faster after exposure to light at 420 nm than at 632 nm. The rate of elimination of PpIX from the volume of detection was faster after 420 nm light irradiation than that after 632 nm. These findings are discussed in view of penetration depths of light and ALA-Me, and diffusion of PpIX.