Summary The mechanism of tumour necrosis photosensitised by liposome-delivered Zn(II) phthalocyanine (Zn-Pc) has been studied in mice bearing a transplanted MS-2 fibrosarcoma. Ultrastructural analyses of tumour specimens obtained at different times after red light-irradiation (300 J cm-2, dose-rate 180 mW cm-2) indicate an early (3 h) photodamage of malignant cells especially at the level of the mitochondria and rough endoplasmic reticulum. The cellular damage becomes more evident between 6 h and 15 h after photodynamic therapy. On the other hand, the capillaries supplying the tumour tissue are modified at a much slower rate and appear to be severely damaged only after 15 h from irradiation, when the whole tissue becomes necrotic. Occasionally, mildly damaged capillaries are observed even at 72 h after irradiation. These findings support the hypothesis that low density lipoproteins (LDL) play a major role in the delivery of Zn-Pc to the tumour tissue; the photosensitiser is released specifically to malignant cells as a consequence of a receptor-mediated endocytosis of LDL.

Summary The covalent binding of a carotene moiety to one phenyl ring and meso-tetraphenyl-substituted porphyrins (see Figure 1) efficiently quenches the photosensitising activity of the porphyrin while a relatively large yield of fluorescence emission around 650 nm is retained. Pharmacokinetic studies performed with two carotenoporphyrins (CPs) and the corresponding porphyrins (Ps) in Balb/c mice bearing an MS-2 fibrosarcoma show that the two Ps give a high selectivity of tumour localisation (tumour/peritumoral tissue ratios of dye concentration ranging between c. 30 and 90 at 24 h after injection of 4.2 -8.4 1tmol kg-in a Cremophor emulsion) and photosensitise tumour necrosis upon red light irradiation. For the same injected doses, the two CPs show no tumour-photosensitising activity even though they localise in the tumour in concentrations of the order of 10-40 jug g-' at 24 h with tumour/peritumoral ratios larger than 10. Thus, the fluorescence emitted by these CPs in the tumour can be used for photodiagnostic purposes with no risk of skin photosensitisation. However, this approach is presently limited by the large accumulation and prolonged retention of the CPs in the liver and spleen.

ZusammenfassungHintergrund und Gegenstand: Im Rahmen der etablierten Tumorbehandlungen hat die Strahlentherapie ihren festen Stellenwert. Auch die Anwendung der photodynamischen Therapie (PDT), insbesondere mit Photofrin II, ist in der Tumorbehandlung bekannt. Chemische Stoffe, die die Wirkung von ionisierenden Strahlen am Tumorgewebe verstärken können, werden bereits in der Medizin genutzt. Keiner dieser Stoffe ist aber ein selektiver Radiosensitizer. Material und Methoden: Mehrere Serien von Tierexperimenten wurden durchgeführt. Nacktmäusen wurden subkutan hochdifferenzierte humane Blasenkarzinom-Zelllinien RT4 implantiert. Den Mäusen wurde 10 mg/kg Photofrin II injiziert, danach wurden sie mit 5 Gy bestrahlt. Ergebnisse: Photofrin II hat sich als chemischer Modulator der biologischen Wirkung ionisierender Strahlen erwiesen. Dies wurde mit der Erhöhung der Tumorverdopplungszeit (Tumorwachstumskurve) von 6,2 auf 10,9 Tage in der Kontrollgruppe bei Anwendung ionisierender Strahlen und Injektion von Porphyrin bewiesen. Schlussfolgerung: Photofrin II zeigt eine gute Wirksamkeit als Radiosensitizer und kann in Zukunft als selektiver Radiosensitizer bei der Tumorbehandlung mit ionisierenden Strahlen genutzt werden. Key Words Radiation therapy · Ionizing irradiation · Photodynamic therapy · Radiosensitizer · Photofrin II Summary Background and Objective:The use of ionizing irradiation as radiation therapy (RT) for tumor treatment represents a well-established method. The use of photodynamic therapy (PDT), especially with Photofrin II, for tumor treatment is also known. Chemical modifiers enhancing the action of radiation therapy are well known and widely used in medicine. None of these compounds, however, is a selective radiosensitizer. Materials and Methods: Several series of animal experiments were performed. The highly differentiated human bladder cancer cell line RT4 was implanted subcutaneously in nude mice. The mice were injected 10 mg/kg Photofrin II and irradiated with 5 Gy. Results: Photofrin II has proved to be a chemical modifier of ionizing irradiation, enhancing the tumor doubling time (tumor growth) from 6.2 to 10.9 days in the control group with the use of irradiation and injection of porphyrin. Conclusion: Photofrin II shows a high activity as radiosensitizer and, in the future, can be used as a selective radiosensitizer for tumor treatment with ionizing radiation.

Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.

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