Enhancement of meta-tetrahydroxyphenylchlorin-sensitized photodynamic treatment on human tumor xenografts using a water-soluble vitamin E analogue, Trolox.

Photodynamic therapy (PDT) using meta-tetrahydroxyphenylchlorin (mTHPC) performed on HT29 human colon adenocarcinoma xenografts in nude mice was shown to be enhanced by Trolox, a water-soluble vitamin E analogue. Trolox, injected i.p. at 250 mg/kg body weight 90 min before PDT, delayed tumor doubling time from 13 (PDT only) to 19 days. Enhancement of the tumoricidal effect of PDT by Trolox required the presence of the drug at the photochemical stage since its injection after irradiation is ineffective. HPLC measurements indicated that 1 hr after injection the Trolox concentration in plasma was as high as 0.8 mM. In vivo measurements of mTHPC fluorescence in mice treated by PDT with or without Trolox injection showed that Trolox did not protect mTHPC from photodegradation. Laser flash photolysis studies performed in solution demonstrated that Trolox reduces triplet mTHPC efficiently (reaction rate constant 2 x 10(7) M(-1) * sec(-1)) leading to the formation of radical products. Kinetic considerations suggest that the Trolox-mediated radical pathway can work in relay with singlet oxygen in hypoxic conditions, providing a possible explanation for the observed enhancement of mTHPC-sensitized PDT by Trolox.

Effects of glutathione peroxidase and catalase on hemolysis and methemoglobin modifications induced by photooxidized psoralen.

Psoralen photooxidation products (POP products) were obtained by UVA irradiation (365 nm, 180-640 W/m2) of an aqueous psoralen solution with fluences of 0-800 kJ/m2. Preincubation of POP products with glutathione peroxidase (GSHPer) or catalase, as well as presence of catalase during UVA irradiation of the aqueous psoralen solution did not influence their hemolytic activity. However, both GSHPer and catalase inhibited POP-induced conversion of methemoglobin. This indicates that hydrogen peroxide and psoralen peroxides destructible by GSHPer, which are being produced during psoralen photooxidation, do not possess hemolytic activity. Furthermore, hydrogen peroxide does not appear to serve as an intermediate in the process of hemolysin formation. Hydrogen peroxide generated during psoralen photooxidation is apparently the main POP product responsible for MetHb conversion.

Photodynamic properties of meta-tetra(hydroxyphenyl)chlorin in human tumor cells.

The photodynamic properties of a second-generation photodynamic sensitizer, meta-tetra(hydroxyphenyl)chlorin (mTHPC) were studied by dye-sensitized photoinactivation (650 nm) of HT29 human adenocarcinoma cells in culture. The photocytotoxicity of mTHPC in vitro depended on the presence of molecular oxygen. A strong inhibition of the photocytotoxicity of mTHPC was observed upon addition of sodium azide, a known singlet oxygen quencher. Photocytotoxicity was not inhibited by scavengers of superoxide anion radical, hydrogen peroxide and hydroxyl radicals. We suggest that mTHPC photosensitizes cell killing predominantly by type II, singlet oxygen-mediated photodynamic reactions. Illumination of cells preloaded with mTHPC induced peroxidation of membrane lipids. Inhibition of photoperoxidation by alpha-tocopherol (0.1 mM) present during illumination did not result in any decrease in toxicity, suggesting that reactions of lipid peroxidation play only a minor role in the overall photocytotoxic effect of mTHPC.

The application of antioxidants in investigations and optimization of photochemotherapy.

Psoralens (furocoumarins) are photosensitizers of plant origin. They are used in combination with near-ultraviolet (320-400 nm) light for the treatment of vitiligo, psoriasis, cutaneous T-cell lymphoma (CTCL), alopecia areata, eczema, and other skin diseases. Photobiological effects of psoralens in humans are numerous. They photosensitize erythema, hyperpigmentation, and skin aging, affect immune system, etc. Molecular mechanisms of photochemical reactions of psoralens with substrates are also numerous. The molecular basis and the relationships between different photobiological effects of psoralens remain the subject of discussion. The use of antioxidants is an essential instrument for clearing up these problems as well as for the progress of photochemotherapy. It was found that antioxidants (alpha-tocopherol, butylated hydroxytoluene, etc.) selectively inhibited the photochemical stage of erythema and hyperpigmentation but had no impact on the post-irradiation stages of these processes. Evidently, the basis of these processes is the reaction of psoralen-photosensitized oxidation of unsaturated lipids and the impairment of barrier functions of biomembranes, since the photochemical stage of these reactions is inhibited by the antioxidant. At the same time, antioxidants did not inhibit the therapeutic effect in the cases of psoriasis and CTCL. Thus, antioxidants can be used as a tool for improvement of psoralen photochemotherapy.