Phthalocyanine-mediated photodynamic therapy induces cell death and a G0/G1 cell cycle arrest in cervical cancer cells.

We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC(50) concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G(0)/G(1) cell cycle arrest during the phthalocyanine PDT-mediated response.

Photodynamic therapy effect of m-THPC (Foscan) in vivo: correlation with pharmacokinetics.

m-Tetra(hydroxyphenyl)chlorin (m-THPC, Foscan, Temoporfin) has an unusually high photodynamic efficacy which cannot be explained by its photochemical properties alone. In vivo interactions are therefore of critical importance in determining this high potency. The pharmacokinetics of m-THPC in a rat tumour model was determined using (14)C m-THPC in an LSBD(1) fibrosarcoma implanted into BDIX rats. The photodynamic therapy (PDT) efficacy was determined at different drug administrations to light intervals and correlated with the tumour and plasma pharmacokinetic data. The plasma pharmacokinetics of m-THPC can be interpreted by compartmental analysis as having three half-lives of 0.46, 6.91 and 82.5 h, with a small initial volume of distribution, suggesting retention in the vascular compartment. Tissues of the reticuloendothelial system showed high accumulation of m-THPC, particularly the liver. PDT efficacy of m-THPC over the same time course seemed to exhibit two peaks of activity (2 and 24 h), in terms of tumour growth delay with the peak at 24 h postinjection correlating to the maximum tumour concentration. Investigation on tumour cells isolated from m-THPC-treated tumours suggested that the peak PDT activity at 2 h represents an effect on the vasculature while the peak at 24 h shows a more direct response. These results indicate that the in vivo PDT effect of m-THPC occurs via several mechanisms.

Porphyrin accumulation induced by 5-aminolaevulinic acid esters in tumour cells growing in vitro and in vivo.

The ability of 5-aminolaevulinic acid and some of its esterified derivatives to induce porphyrin accumulation has been examined in CaNT murine mammary carcinoma cells growing in culture and as tumours in vivo. Topical or intravenous administration of 5-aminolaevulinic acid-esters to mice bearing subcutaneous tumours produced lower porphyrin levels in the tumour than an equimolar dose of 5-aminolaevulinic acid. Reducing the dose of intravenous hexyl- or benzyl-ALA and topical hexyl-5-aminolaevulinic acid resulted in a dose-dependent reduction in porphyrin accumulation. A number of normal tissues accumulated higher concentrations of porphyrins than tumour tissue following intravenous administration of 5-aminolaevulinic acid-esters. Esterase activity in these normal tissues was greater than that in tumour tissue. In contrast to the situation in vivo, all of the 5-aminolaevulinic acid-esters examined were at least as effective as 5-aminolaevulinic acid when applied to cloned CaNT cells in vitro, with the drug concentration required for maximum porphyrin accumulation varying with ester chain-length. Tumour cells growing in culture released esterase activity into the medium. These findings suggest that the efficacy of 5-aminolaevulinic esters may vary depending on the esterase activity of the target tissue, and suggest caution when interpreting the findings of in vitro studies using these and similar prodrugs.

Investigation of cross-resistance to a range of photosensitizers, hyperthermia and UV light in two radiation-induced fibrosarcoma cell strains resistant to photodynamic therapy in vitro.

Two distinct photodynamic therapy-resistant variants of the murine radiation-induced fibrosarcoma (RIF) cell line have been isolated. One strain displayed relative resistance over the parental RIF-1 strain to treatment with the porphyrin-based compound, polyhaematoporphyrin (PHP), whereas the other strain displayed relative resistance over the RIF-1 strain to treatment using the cationic zinc (II) pyridinium-substituted phthalocyanine (PPC). The PHP-resistant strain did not display cross-resistance to PPC-mediated treatment, and vice versa. In both PDT-resistant strains, the increased resistance could not be attributed to altered cellular growth rate, antioxidant capacity or intracellular sensitizer localization. The PHP-resistant strain displayed resistance to treatment with both short (1 h) and extended (16 h) sensitizer incubation periods, which may indicate that in this strain, the resistance has arisen through an alteration in a membrane component. Conversely, the PPC-resistant strain only displayed increased resistance over the parental cells to treatment involving the short drug incubation, which is likely to reflect the existence of a threshold effect caused by the alteration of an individual cellular target. Each resistant strain has been compared to the parental strain in terms of cellular sensitivity to treatment with a range of other photosensitizers, hyperthermia, UV light and the anticancer agent cis-diamminedichloroplatinum. The PHP-resistant strain exhibited crossresistance to photosensitization treatment using exogenously added protoporphyrin IX, and also to treatment with the anionic phthalocyanine sensitizers, zinc (II) tetrasulfonated phthalocyanine and zinc (II) tetraglycine-substituted phthalocyanine. The PPC-resistant strain did not display cross-resistance to any of the treatment strategies employed in this investigation. The results of this investigation indicate that there are at least two distinct mechanisms of PDT resistance in RIF cells, and that the mechanism of PHP resistance may, to some extent depend, upon the physical nature of the sensitizer molecule.

Decreased efficiency of trypsinization of cells following photodynamic therapy: evaluation of a role for tissue transglutaminase.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cellular damage are to be fully understood. The relationship between sensitizer, fluence rate and the removal of cells by trypsinization was studied using the RIF-1 cell line. Following treatment of RIF-1 cells with pyridinium zinc (II) phthalocyanine (PPC), or polyhaematoporphyrin at 10 mW cm-2 (3 J cm-2), there was a significant number of cells that were not removed by trypsin incubation compared to controls. Decreasing the fluence rate from 10 to 2.5 mW cm-2 resulted in a two-fold increase in the number of cells attached to the substratum when PPC used as sensitizer; however, with 5,10,15,20 meso-tetra(hydroxyphenyl) chlorine (m-THPC) there was no resistance to trypsinization following treatment at either fluence rate. The results indicate that resistance of cells to trypsinization following PDT is likely to be both sensitizer and fluence rate dependent. Increased activity of the enzyme tissue-transglutaminase (tTGase) was observed following PPC-PDT, but not following m-THPC-PDT. Similar results were obtained using HT29 human colonic carcinoma and ECV304 human umbilical vein endothelial cell lines. Hamster fibrosarcoma cell (Met B) clones transfected with human tTGase also exhibited resistance to trypsinization following PPC-mediated photosensitization; however, a similar degree of resistance was observed in PDT-treated control Met B cells suggesting that tTGase activity alone was not involved in this process.

Improved response of plaque psoriasis after multiple treatments with topical 5-aminolaevulinic acid photodynamic therapy.

We investigated the clinical response of 10 patients with plaque psoriasis to multiple treatments with photodynamic therapy, using topical application of 5-aminolaevulinic acid followed by exposure to broad-band visible radiation. Treatment was performed up to 3 times per week, with a maximum of 12 treatments, using a light dose of 8 Jcm(-2) delivered at a dose-rate of 15 mW cm(-2). Eight patients showed a clinical response. Out of 19 treated sites, 4 cleared, 10 responded but did not clear and 5 showed no improvement. Of the 4 sites that cleared only 1 did so fully, after 7 treatments, 45 days after the start of therapy. Of the 10 sites that responded partially, the greatest reduction in scale, erythema and induration index occurred after a minimum of 3 and a maximum of 8 treatments. The intensity of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence, recorded prior to the first treatment, varied between sites on the same patient as well as between patients. There was also a variation in fluorescence intensity recorded from the same site immediately prior to subsequent treatments, although the pretreatment levels generally decreased as the study progressed and then increased as psoriasis relapsed. Biopsies confirmed that fluorescence was localized throughout the epidermis and stratum corneum, but the level was not consistent between sections taken within the same biopsy. We also observed fluorescence at sites distant from the ones that received 5-aminolaevulinic acid, which was not present prior to the start of the treatment programme, but found no evidence of elevated levels of plasma porphyrins. The level of discomfort associated with this therapy increased with increasing values of the calculated photodynamic dose, defined as the product of the initial photosensitizer concentration and the percentage reduction in fluorescence following irradiation. Therefore, although clinical efficacy improved with multiple treatments, unpredictable response and patient discomfort make ALA-PDT unsuitable for the treatment of psoriasis.

The induction of apoptosis by a positively charged methylene blue derivative.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cell death are to be fully understood. PDT with a methylene blue analog DO15 yielded mitochondrial photodamage whilst membrane and lysosomal integrity were maintained. Apoptosis was detected using the DNA stain HO342, by the appearance of 50 kb fragments and by DNA ladder formation. The release of mono- and oligonucleosomes was further quantified using an ELISA protocol. Large DNA fragments were observed immediately following illumination, and nucleosomes were detected at 1-2 h post-treatment. Increasing the dose 4-fold accelerated the apoptotic response to PDT. This is the first report of a thiazine photosensitiser inducing apoptosis and is consistent with recent proposals suggesting that release of mitochondrial components may play an important role in the mechanism of cell death.

Photoinactivation of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate both gram-negative and gram-positive bacteria.

The photosensitization of microorganisms is potentially useful for sterilization and for the treatment of certain bacterial diseases. Until now, any broad spectrum approach has been inhibited because, although Gram-positive bacteria can be photoinactivated by a range of photosensitizers, Gram-negative bacteria have not usually been susceptible to photosensitized destruction. In the present work, it has been shown that the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as the Gram-positive bacterium Enterococcus seriolicida, can be photoinactivated when illuminated in the presence of a cationic water-soluble zinc pyridinium phthalocyanine (PPC). The degree of photoinactivation is dependent on both the concentration of PPC and the illumination time. In contrast, the three bacteria are not photoinactivated by illumination in the presence of a neutral tetra-diethanolamine phthalocyanine (TDEPC) or negatively charged tetra-sulphonated phthalocyanine (TSPC). Uptake studies have revealed that the lack of activity of TSPC is due to the fact that it has very little affinity for any of the organisms. However, the issue appears to be more complex than simply the gross levels of cellular uptake, since TDEPC and PPC are both taken up by the organisms but only PPC shows activity. This indicates that the localization and subcellular distribution of the phthalocyanines may be a crucial factor in determining their cell killing potential. Further analysis of the uptake data has revealed a cell-bound photosensitizer fraction, which remains tightly associated after several washings, and another weakly bound fraction, which is removed by successive washings. Analysis of the cell killing curves, carried out after successive washings of E. coli exposed to PPC, has revealed that it is the tightly associated fraction that is involved in the photosensitization. Taken together with other data, these results suggest that cationic photosensitizers may have a broader application in the photoinactivation of bacterial cells than the anionic or neutral photosensitizers commonly used in photodynamic therapy.

Photosensitization of the endometrium with topical 5-aminolevulinic acid.

Photosensitization of the endometrium was attempted in vitro and in vivo by intrauterine administration of 5-aminolevulinic acid, which is converted to the photosensitizer protoporphyrin IX. Protoporphyrin IX was found in both functional and basal layers of the endometrial glands at concentrations nine and 10 times higher than myometrium in in vitro and in vivo experiments, respectively. Selective endometrial photosensitization is possible with topical 5-aminolevulinic acid, but it might not be distributed sufficiently evenly for use as a sensitizing agent in photodynamic ablation.

Mouse skin photosensitivity with dihaematoporphyrin ether (DHE) and aluminium sulphonated phthalocyanine (AlSPc): a comparative study.

Skin photosensitivity of sun exposed sites is the major side effect of dihaematoporphyrin ether (DHE) photodynamic therapy (PDT). Reports of severe oedema and erythema have generally been anecdotal. We have studied aluminium sulphonated phthalocyanine (AlSPc) as a potential photosensitiser for PDT. In this paper we report our work comparing the skin photosensitivity reactions of DHE and AlSPc. We have studied: (i) the time course of the skin reactions, (ii) the effect of increasing time from administration of photosensitiser to irradiation, (iii) drug-skin reaction dose response. Groups of Skh I female hairless albino mice were given an intravenous bolus dose of either 0.9% saline solution, AlSPc or DHE (Photofrin II). Drug doses ranged from 0.5 to 50 mg/kg. At times ranging from 1 h to 1 month animals were irradiated with a range of doses of solar simulated radiation (SSR). The skin reaction was observed over a 2 week period. DHE reactions were always more severe than those with AlSPc. Peak skin reaction was seen at 3 h for DHE and 6 h for AlSPc. DHE reactions were still visible 2 weeks after irradiation whereas the AlSPc reaction disappeared by 48 h. Irradiation evoked a reaction up to 2 months after administration of DHE but only up to 2 weeks with AlSPc. The mean SSR dose at which a skin reaction was seen decreased with increasing dose of both agents. The rate of decrease was slower with AlSPc than DHE. This study suggests that in PDT, AlSPc will cause much less skin photosensitivity than DHE.