David I. Vernon

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.

Mechanism of uptake of a cationic water-soluble pyridinium zinc phthalocyanine across the outer membrane of Escherichia coli

ABSTRACT Previous studies have shown that a cationic water-soluble pyridinium zinc phthalocyanine (PPC) is a powerful photosensitizer that is able to inactivate Escherichia coli. In the current work incubation of E. coli cells with PPC in the dark caused alterations in the outer membrane permeability barrier of the cells, rendering the bacteria much more sensitive to hydrophobic compounds, with little effect seen with hydrophilic compounds. Addition of Mg2+ to the medium prior to incubation of the cells with PPC prevented these alterations in the outer membrane permeability barrier. The presence of Mg2+ in the medium also prevented the photoinactivation of E. coli cells with PPC. These results are consistent with the hypothesis that PPC gains access across the outer membrane of E. coli cells via the self-promoted uptake pathway, a mechanism of uptake postulated for the uptake of other cationic compounds across the outer membranes of gram-negative bacteria.

In Vitro Photodynamic Activity of a Series of Methylene Blue Analogues

We have synthesized a series of symmetrical phenothiazines in which the methyl groups of methylene blue have been substituted by longer alkyl chains. Intrinsic photosensitizing ability was not altered by increasing the chain length. However, in vitro phototoxicity after 2 h incubation of RIF‐1 murine fibrosarcoma cells followed the order n‐propyl > n‐pentyl > n‐butyl > n‐hexyl > ethyl > methyl, with ethyl and n‐propyl analogues being 14‐ and 130‐fold more phototoxic than methylene blue, respectively. All analogues also had an improved ratio of phototoxicity : dark toxicity (4:1 to 27:1) compared with methylene blue (3:1). Phototoxicity did not correlate with cellular phenothiazine levels, suggesting that the site of subcellular localization may be more important. After 2 h incubation of RIF‐1 cells with the phototoxicity LD50 concentration, methylene blue and all analogues were observed to be localized in the lysosomes by fluorescence microscopy. On exposure to light, methylene blue relocalized to the nucleus, the ethyl analogue did not relocalize, whereas the more phototoxic n‐propyl –n‐hexyl analogues relocalized to the mitochondria. Relocalization to the mitochondria was associated with an octanol : buffer partition coefficient ≥ 1. Therefore, the longer‐chain analogues of methylene blue show significantly improved phototoxicity in vitro and, in addition, are expected to avoid the problems of mutagenicity associated with the nuclear localization of methylene blue.

Endogenous porphyrin distribution induced by 5-aminolaevulinic acid in the tissue layers of the gastrointestinal tract

The accumulation of endogenous porphyrins in rats following systemic administration of 5-aminolaevulinic acid (ALA) has been examined to assess the photosensitization characteristics of this technique for photodynamic therapy (PDT) and chemical extraction assays with fluorescence and absorbance detection of the porphyrin content have been carried out. We compared the results obtained using quantitative microfluorimetry on normal gastric and colonic tissues in rats at 0.5, 1, 2, 4 and 6 h and chemically induced duodenal tumours 2 and 4.5 h after intravenous administration of ALA at a dose of 200 mg kg-1. With chemical extraction followed by high performance liquid chromatography analysis, protoporphyrin IX (PpIX) was found to be the predominant porphyrin present, reaching peak levels of several microgrammes per gramme at 2-4 h in each type of tissue; a small amount of coproporphyrin was detected at 0.5 and 2 h in normal gastric mucosa and duodenal tumour respectively. Both the extraction assay and quantitative microfluorimetry showed that the porphyrin fluorescence builds up rapidly in the mucosal layers of the colon and stomach, reaching a maximum at 2 h, whereas lower fluorescence levels were found with a slower rate of accumulation in the corresponding muscularis layers. A significant PpIX content was found in the duodenal tumour, with a maximum of 7.1 micrograms g-1 4.5 h after ALA administration. We conclude that systemic administration of ALA can induce effective tissue sensitization with protoporphyrin IX and appears to be a promising technique for PDT.

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.

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 14C m-THPC in an LSBD1 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.

5-Aminolevulinic acid photosensitization of dysplastic Barrett's esophagus: a pharmacokinetic study.

Photodynamic therapy (PDT) using 5‐aminolevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) may have a role in the treatment of dysplastic Barretts esophagus. Before ALA‐induced PDT can be used clinically, optimum treatment parameters must be established. In this study of 35 patients, the issues of drug dosage, time interval between drug and light delivery and side effects of oral ALA administration are addressed. Spectrofluoro‐metric analysis of tissue samples demonstrates that oral ALA administration induces porphyrin accumulation in esophageal tissues, with maximum levels at 4–6 h. High‐performance liquid chromatography confirms the identity of this porphyrin as PpIX, and fluorescence microscopy analysis demonstrates that it preferentially accumulates in the esophageal mucosa, rather than in the underlying stroma. Side effects of ALA administration included malaise, headache, photosensitivity, alopecia, transient derangement of liver function, nausea and vomiting. Fewer side effects and less hepatic toxicity was seen with 30 mg/kg than 50 mg/kg ALA. In conclusion, oral ALA administration induces preferential PpIX accumulation in the esophageal mucosa, with peak PpIX fluorescence noted at 4 h and minimal systemic toxicity at a dose of 30 mg/kg.

MOUSE SKIN PHOTOSENSITIVITY WITH DIHAEMATOPORPHYRIN ETHER (DHE) AND ALUMINIUM SULPHONATED PHTHALOCYANINE (AlSPc)

Abstract 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.

5-Aminolaevulinic acid methyl ester transport on amino acid carriers in a human colon adenocarcinoma cell line.

Abstract The transport mechanisms of 5-aminolevulinic acid methyl ester (5-ALA-ME) have been studied in a human adenocarcinoma cell line (WiDr) by means of 14[C]-labeled 5-ALA-ME. The transport was found to be partly Na+ dependent, while the extracellular Cl− concentration did not affect the uptake. The transport of 5-ALA-ME into WiDr cells was dependent on the incubation temperature and was found to be completely blocked by the inhibitors of energy metabolism, 2-deoxyglucose and sodium azide. WiDr cells were treated with 10 mM of 14 different amino acids and the substrate specificity of the 5-ALA-ME transporter(s) was analyzed by treating the cells with 23 μM or 1 mM 14[C]-labeled 5-ALA-ME. The transport of 5-ALA-ME was found to be inhibited to the highest extent, i.e. about 60%, by the nonpolar amino acids l-alanine, l-methionine, l-tryptophan and glycine. The uptake of 5-ALA-ME followed an exponential decay with increasing concentration of glycine, reaching a maximum inhibition of uptake of 5-ALA-ME of 55%. Sarcosine, a specific inhibitor of system Gly, did not significantly inhibit 5-ALA-ME transport. In contrast to transport of 5-ALA, 5-ALA-ME does not seem to be taken up by system BETA transporters. In conclusion, the cellular uptake of 5-ALA-ME into WiDr cells seems to be due to active transport mechanisms, involving transporters of nonpolar amino acids.

A Comparative Study of the Cellular Uptake and Photodynamic Efficacy of Three Novel Zinc Phthalocyanines of Differing Charge

Abstract— Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20‐tetra(m‐hydroxyphenyl)chlorin (m‐THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF‐1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly unproved activity over PHP and m‐THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m‐THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low‐level light exposure, and this was charge dependent The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zhic [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low‐level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.