aluminum-phthalocyanine-disulfonate and Necrosis

Photochemical internalisation (PCI) is a delivery technology that employs a sub-lethal form of photodynamic therapy (PDT) in which a photosensitiser is activated by light to break down intracellular membranes and release macromolecules into the cytosol where they can be biologically active. Although PCI does enhance the PDT killing of transplanted tumours in mice after local injection of the cytotoxic agent, gelonin, the redistribution of gelonin from intracellular organelles into the cytosol has only previously been demonstrated in vitro. This study is designed to understand the factors controlling the efficacy of PCI in vivo and to document the mechanism of action. Using the photosensitiser AlS(2)Pc in studies on normal rat liver, we have demonstrated in vivo that gelonin is initially taken up into lysosomes, but can be released into the cytosol using PCI. Furthermore, PCI enhances the PDT effect after systemic administration of gelonin (volume of necrosis increased x2.5 when gelonin is given one hour before light), with the remarkably low dose of 5 microg/kg (10,000 times lower than the LD50); in the absence of light, there is no effect with 500 microg/kg. These results suggest that PCI may have a useful role to play in the site specific activation of cytotoxic agents like gelonin, given at a dose level that has no effect in the absence of light.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cytosol; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Indoles; Light; Liver; Lysosomes; Necrosis; Organometallic Compounds; Photochemical Processes; Photochemotherapy; Photosensitizing Agents; Rats; Rats, Wistar; Ribosome Inactivating Proteins, Type 1

Photodynamic therapy (PDT) requires a photosensitising drug, light and oxygen. While it is known that the haemoglobin oxygen saturation (HbSat) can be altered by PDT, little has been done to correlate this with microvascular changes and the final biological effect. This report describes such studies on the normal liver of rats sensitised with aluminium disulphonated phthalocyanine. In total, 50 J of light at 670 nm, continuous or fractionated at 25 or 100 mW, was applied with a single laser fibre touching the liver surface. HbSat was monitored continuously 1.5-5.0 mm from the laser fibre using visible light reflectance spectroscopy (VLRS). Vascular shutdown was assessed by fluorescein angiography 2-40 min after light delivery. Necrosis was measured at post mortem 3 days after PDT. In all treatment groups at a 1.5 mm separation, HbSat fell to zero with little recovery after light delivery. At 2.5 mm, HbSat also decreased during light delivery, except with fractionated light, but then recovered. The greatest recovery of fluorescein perfusion after PDT was seen using 25 mW, suggesting an ischaemia/reperfusion injury. Necrosis was more extensive after low power and fractionated light than with 100 mW, continuous illumination. We conclude that VLRS is a useful technique for monitoring HbSat, although the correlation between HbSat, fluorescein exclusion and necrosis varied markedly with the light delivery regimen used.

Topics: Animals; Female; Fluorescein Angiography; Hemoglobins; Indoles; Ischemia; Liver; Necrosis; Organometallic Compounds; Oxygen; Perfusion; Photochemotherapy; Photosensitizing Agents; Rats; Rats, Wistar; Spectrum Analysis

The use of photodynamic therapy (PDT) for the treatment of skin and oral cancer has been the subject of several clinical studies but there has been little scientific evaluation of its mechanism of action. Evidence to date suggests that whilst epithelial cell death may be secondary to vascular damage, direct cell killing may occur and may involve an apoptosis-like mechanism. To investigate the mechanism of epithelial cell death following PDT, two cell lines, human epidermal keratinocytes (UP) and oral squamous cell carcinoma-derived cells (H376) were subjected to PDT with aluminium disulphonated phthalocyanine (AlS2Pc) as the photosensitizer and red laser light at 675 nm. Control groups received red laser light, photosensitizer or neither. The effects of PDT were assessed using an MTS cell-proliferation assay, which showed a significant reduction in viability (p < 0.01) for PDT-treated cells compared to controls. For morphological analysis, cells were stained with haemotoxylin and eosin and the numbers showing typical apoptotic features counted. The treated cultures showed significantly increased numbers of apoptotic cells. Moreover, the H376 control cultures showed a baseline level of apoptosis of approx. 15%. Apoptosis was confirmed by ultrastructural analysis and by in situ end-labeling of DNA fragments. The results show that PDT using AlS2Pc as a photosensitizer promotes apoptotic cell death in UP and H376 cells in vitro and suggest that direct killing of epithelial cells may contribute to tumour necrosis in vivo.

Topics: Aluminum; Apoptosis; Carcinoma, Squamous Cell; Cell Count; Cell Division; Cell Line; Cell Survival; Coloring Agents; DNA Fragmentation; Eosine Yellowish-(YS); Epidermis; Epithelial Cells; Fluorescent Dyes; Hematoxylin; Humans; Indoles; Keratinocytes; Laser Therapy; Mouth Neoplasms; Necrosis; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Tumor Cells, Cultured

Surgery is the only effective treatment for dysplasia in the gastrointestinal tract with considerable associated morbidity and mortality and is difficult to justify without confirmed malignancy. Photodynamic therapy (PDT) produces localised necrosis, which can be limited to the mucosa. This study examined the mechanical properties of the normal rat stomach after PDT. The aim of this study was to measure the bursting pressure of PDT lesions in the stomach and to assess gastric emptying after producing circumferential mucosal necrosis at the pylorus by PDT. Two photosensitising agents were used--5-aminolaevulinic acid (ALA), and aluminium disulphonated phthalocyanine (A1S2Pc). Normal rats were sensitised and PDT lesions created in the stomach with red light. The bursting pressure was measured and compared with that in thermal control lesions. In further experiments, circumferential mucosal necrosis was produced at the pylorus, and animals observed for subsequent eating and weight gain. It was found that gastric bursting pressure was reduced after thermal injury, but not at any time after PDT (with A1S2Pc, but not ALA, adhesive omental reinforcement was required to maintain the gastric wall strength at one week). For the pyloric lesions, gastric emptying was permanently impaired using A1S2Pc, but with low dose ALA (20 mg/kg) had returned to normal by three days. With ALA, but not A1S2Pc, necrosis could be limited to the mucosa. In conclusion, using ALA, selective ablation of the gastric mucosa is possible, which does not reduce the strength of the stomach and only temporarily delays gastric emptying. PDT is a promising technique for the circumferential ablation of dysplastic mucosa.

Topics: Aminolevulinic Acid; Animals; Female; Gastric Emptying; Gastric Mucosa; Indoles; Necrosis; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Pressure; Rats; Rats, Wistar; Rupture

We have previously reported photodynamic therapy of normal rat colon using aluminium sulphonated phthalocyanine (AISPc). In that study, the AISPc used was a mixture of phthalocyanines of different degrees of sulphonation. Phthalocyanines of defined degrees of sulphonation have recently become available and we compared the distribution of the di- and tetra-sulphonates (AIS2Pc and AIS4Pc) in rat colon and colon wall structures employing both chemical extraction and fluorescence photometry using a charge coupled device imaging system. Also, the photodynamic effects produced by these components in rat colon were compared at various times after photosensitization. After intravenous photosensitizer administration using equimolar doses, the concentration of AIS2Pc in colon fell off more rapidly with time than AIS4Pc. Differences were noted in the microscopic distribution of these compounds, with the di-sulphonate exhibiting peak fluorescence in colon wall structures by 1 h after photosensitization, while mucosal fluorescence with the tetra-sulphonate peaked at 5 h. Fluorescence was also lost from the colon wall much more slowly with the tetra-sulphonate, which tended to be retained in the submucosa. Maximum photosensitizing capability was seen at 1 h with AIS2Pc and no lesions could be produced with photodynamic therapy at 1 week, with up to 5.65 mumol/kg. With AIS4Pc (5.65 mumol/kg), while no lesions could be produced with light treatment at 1 h, photodynamic therapy at 1 week produced lesions only slightly smaller than those produced with treatment at 48 h (the time of maximum effect), and significant photosensitization was present at 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aluminum; Animals; Colon; Female; Indoles; Necrosis; Organometallic Compounds; Photochemotherapy; Radiation-Sensitizing Agents; Rats; Rats, Inbred Strains