Dougherty, 1979). After 24-72h, to allow clearance of the photosensitiser from normal tissues, the malignant lesions are exposed to visible light, usually by laser irradiation. Tumour necrosis and regression ensue from photoradiation. It is generally agreed that cytotoxicity is mediated via formation of the highly reactive oxygen species, singlet oxygen, 102, (Weishaupt et al., 1976;Gibson et al., 1984;Parker, 1987). Since the original promising clinical results utilised HpD, a crude preparation composed of at least seven different porphyrin species (Gibson et al., 1984; Kessel, 1986;Moan et al., 1987), subsequent investigations were directed towards determination of the chemical structure of the 'active component' of HpD (Moan et al., 1982;Kessel and Chou, 1983;Dougherty et al., 1984). Methods developed to purify HpD produced a porphyrin mixture enriched in the hydrophobic components, reported to be mainly dihaematoporphyrin ethers or esters (Byrne et al., 1987;Dougherty, 1987;Kessel et al., 1987). This enriched preparation, Photofrin II, is now commercially produced for clinical and laboratory studies.In our earlier studies we utilised HpD as the photosensitiser (Hilf et al., , 1984Gibson et al., 1984). Because of the complex nature of HpD, the intracellular localisation and effects of various photosensitising components relative to time after administration could differ from the pharmacokinetics that would be observed with Photofrin II. We therefore undertook a study of Photofrin II employing the same in vivo-in vitro protocol used previously for HpD (Hilf et al., 1984). In this protocol, the photosensitiser is injected into tumour-bearing animals, tumours are removed and subcellular organelles are prepared. These preparations are then exposed to visible light in vitro, and various biochemical endpoints are analysed, such as site-specific enzyme activities. One advantage of this protocol is that it takes into account any metabolism of the sensitiser by the tumour-bearing host. In this report, using Photofrin II as the photosensitiser, data are presented on the time-course and drug dose response of photosensitisation of selected mitochondrial and cytosolic enzymes in the R3230AC mammary carcinoma. Materials and methods MaterialsPhotofrin II was kindly provided by Photomedica Inc., Raritan, NJ. All other reagents were obtained from Sigma Chemical Co., St Louis, MO, unless otherwise noted. Animals and tumoursThe R3230AC mammary adenocarcinoma was maintained by subcutaneous transplantation in the axillary region of 60-80g female Fischer rats, using the sterile trochar procedure described previously (Hilf et al., 1965).Preparation of subcellular organelles from tumours For in vitro studies, tumour-bearing rats were killed 17-24 days after implantation of the R3230AC mammary adenocarcinoma. From excised tumours, mitochondria were prepared according to methods described earlier . Briefly, R3230AC tumours were removed, weighed, placed in a dish on ice in 0.9% NaCl solution and minced with scissors. Approximately 2g...

Summary Photodynamic therapy consists of the systemic administration of a derivative of haematoporphyrin (Photofrin 11) followed 24-72 h later by exposure of malignant lesions to photoradiation. We investigated the efficacy of this treatment after direct intratumoral injection of Photofrin II. This direct treatment regimen resulted in higher rates of inhibition of mitochondrial cytochrome c oxidase (5.13% J' cmM2 x 10-') and succinate dehydrogenase (3.14% J-' cm-2 X 10-I) in vitro at 2 h after intratumoral injection compared to rates of inhibition obtained after intraperitoneal drug administration: 0.51 and 0.42% J -I cm-2 x 10-', respectively. A significant delay in tumour growth in vivo was observed in animals that received intratumoral injections 2 h before photoradiation compared to animals injected intraperitoneally at either 2 or 24 h before photoradiation. The treatment protocols were compared with control groups, consisting of Photofrin II administration intratumorally or intraperitoneally without photoradiation, or photoradiation in the absence of Photofrin II. These data indicate that the intratumoral injection regimen with Photofrin II enhanced the efficacy of photodynamic therapy. The greater delay in tumour growth observed after intratumoral administration of Photofrin II suggests a mechanism favouring direct cell damage.