Stanley B. Brown

The present and future role of photodynamic therapy in cancer treatment.

It is more than 25 years since photodynamic therapy (PDT) was proposed as a useful tool in oncology, but the approach is only now being used more widely in the clinic. The understanding of the biology of PDT has advanced, and efficient, convenient, and inexpensive systems of light delivery are now available. Results from well-controlled, randomised phase III trials are also becoming available, especially for treatment of non-melanoma skin cancer and Barretts oesophagus, and improved photosensitising drugs are in development. PDT has several potential advantages over surgery and radiotherapy: it is comparatively non-invasive, it can be targeted accurately, repeated doses can be given without the total-dose limitations associated with radiotherapy, and the healing process results in little or no scarring. PDT can usually be done in an outpatient or day-case setting, is convenient for the patient, and has no side-effects. Two photosensitising drugs, porfirmer sodium and temoporfin, have now been approved for systemic administration, and aminolevulinic acid and methyl aminolevulinate have been approved for topical use. Here, we review current use of PDT in oncology and look at its future potential as more selective photosensitising drugs become available.

Guidelines for topical photodynamic therapy: report of a workshop of the British Photodermatology Group

Summary Topical photodynamic therapy (PDT) is effective in the treatment of certain non‐melanoma skin cancers and is under evaluation in other dermatoses. Its development has been enhanced by a low rate of adverse events and good cosmesis. 5‐Aminolaevulinic acid (ALA) is the main agent used, converted within cells into the photosensitizer protoporphyrin IX, with surface illumination then triggering the photodynamic reaction. Despite the relative simplicity of the technique, accurate dosimetry in PDT is complicated by multiple variables in drug formulation, delivery and duration of application, in addition to light‐specific parameters. Several non‐coherent and coherent light sources are effective in PDT. Optimal disease‐specific irradiance, wavelength and total dose characteristics have yet to be established, and are compounded by difficulties comparing light sources. The carcinogenic risk of ALA‐PDT appears to be low. Current evidence indicates topical PDT to be effective in actinic keratoses on the face and scalp, Bowens disease and superficial basal cell carcinomas (BCCs). PDT may prove advantageous where size, site or number of lesions limits the efficacy and/or acceptability of conventional therapies. Topical ALA‐PDT alone is a relatively poor option for both nodular BCCs and squamous cell carcinomas. Experience of the modality in other skin diseases remains limited; areas where there is potential benefit include viral warts, acne, psoriasis and cutaneous T‐cell lymphoma. A recent British Photodermatology Group workshop considered published evidence on topical PDT in order to establish guidelines to promote the efficacy and safety of this increasingly practised treatment modality.

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.

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.

Photosensitized inactivation of microorganisms

Despite major advances in medicine in the last 100 years, microbiologically-based diseases continue to present enormous global health problems. New approaches that are effective, affordable and widely applicable and that are not susceptible to resistance are urgently needed. The photodynamic approach is known to meet at least some of these criteria and, with the creation and testing of new photosensitisers, may develop to meet all of them. The approach, involving the combination of light and a photosensitising drug, is currently being applied to the treatment of diseases caused by bacteria, yeasts, viruses and parasites, as well as to sterilisation of blood and other products.

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.

The distribution of porphyrins with different tumour localising ability among human plasma proteins

The distribution among the main fractions of human plasma lipoproteins of a number of porphyrins with different tumour localising ability has been determined by means of ultracentrifugation. A main trend is that the fraction of the dyes that are bound to low density lipoprotein (LDL) increases, and the fraction bound to HSA decreases with decreasing polarity of the dyes. An asymmetric charge distribution, such as in TPPS2a, favours LDL-binding more than expected on the basis of lipophilicity. No correlation between the known tumour localising ability of the drugs tested in the present work and their relative affinity for LDL was found. One of the best tumour localisers reported in the literature, TPPS4, hardly binds to LDL, while Hp and Pp, which are commonly considered inefficient tumour localisers, do have a significant affinity for LDL. On the other hand, the LDL binding capacity for a drug is suggested to be a good index for cellular uptake. Such an index does not necessarily imply that the actual uptake occurs by the LDL pathway.

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.

The place of bronchoscopic photodynamic therapy in advanced unresectable lung cancer: experience of 100 cases

OBJECTIVES The objectives of the study were: (1) to evaluate effectiveness of photodynamic therapy (PDT) for symptom palliation in patients with inoperable lung cancer; (2) to determine survival benefit in a subset of patients. METHODS One hundred patients, 68 male, 32 female, aged 44-81 years (mean 62.5) with advanced inoperable bronchogenic cancer and endobronchial luminal obstruction were prospectively studied. Eighty-two percent had previous chemo/radiotherapy. The pre-treatment protocol consisted of: clinical, radiological and bronchoscopic examination, pulmonary function testing, assessment of WHO performance status and clinical staging. Treatment protocol was: intravenous injection of 2 mg/kg body weight of photofrin/polyhaematoporphyrin and interstitial illumination using 630 nm laser light 24-72 h later. Follow-up was at 6-8 weeks for 1 year. Then every 3-6 months if applicable. Repeat PDT as necessary. RESULTS All patients were stage IIIa-IV. The histology of the tumour was: non small cell in 90 and small cell in 10. There was no treatment related mortality. Mean endoluminal obstruction fell from 85.8% to 17.5%, mean forced vital capacity (FVC) and forced expiratory volume in 1s (FEVI) improvement was 430 ml and 280 ml, respectively. Ninety patients died from 6 weeks to 37 months, mean and median survival: 9 months and 5 months, respectively. Ten patients are alive from 13 to 72 months, mean 36 months, median 29 months. Overall 2-year survival was 19%. Multivariant analysis indicated that age, sex, histology and stage of disease did not influence survival significantly but performance status did. Patients with WHO < 2 had mean and median survival of 17.8 and 14 months versus WHO > 2, 6.9 mean and 4 months median survival (log-rank P < 0.0001). CONCLUSIONS (1) PDT is effective in palliation of inoperable advanced lung cancer. (2) Subset of patients with a better performance status have added survival benefit.

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.