Skin Phototoxicity

This section will briefly focus on tests used to identify chemicals that lead to toxic responses after contact with skin and a subsequent exposure to light. This involves locally as well as systemically administered substances. 

This test enables to identify compounds which show a phototoxic effect in vivo 

The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (-UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is assessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) one day after the initial treatment. Normally, healthy cells may incorporate and bind NR. Alterations of the cell surface or the lysosomal membranes, however, lead to a decreased uptake and binding of the dye. 

For the in vitro test, the fibroblasts are allowed to form a half-confluent monolayer within 24 h. Different concentrations of the test chemical are then incubated for 1 h with two sets of cells in parallel (typically on 96-well plates, 104 cells per well, passage number 100). After the incubation with the test substances, one set is irradiated with a nontoxic dose of UVA light (5 J/cm2), while the other set is kept in the dark. Twenty hours after irradiation, cell viability is evaluated by measuring the uptake of NR for 3 h. After the end of the absorption process, excess NR is removed and the cells are treated with an NR desorption solution (ethanol/acetic acid) to extract the dye taken up by the cells. Subsequently, the optical density of the NR solution is measured at 540 nm. As positive control, a test with chlorpromazine is performed. 

Reconstructed Human Epidermis in Combination with MTT-Assay 

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Photofrin (QLT) 628 (3000) 0.89 1.2-5 75-250 750-2500 Early treatment of bladder, lung, esophagus, cervix, stomach and mouth Palliative in later stages Skin phototoxicity (up to 4 weeks) is a problem (Approved, esophegal and lung)... 

BPD (QLT) Visudyne (Cibavision) 690 (34 000) 0.84 4 150 1500 Skin cancer Age-related macular degeneration No skin phototoxicity. Difficult synthesis (Approved, age-related macular degeneration)... 

NPe6 Nippon Lab 660 (40 000) 0.77 1.0 25-200 1500 Skin cancers Lung No skin phototoxicity... 

Lu-Tex Lutrin (Phannacyclics) 732 (42 000) 0.56 1.0 150 1500 Breast cancer, malignant melanoma, basal cell carcinoma No skin phototoxicity Pain associated in skin during light treatment... 

ALA-proto-porphyrin IX Levulan (DUSA) 635 (5000) 0.56 60 50-150 500-1500 Skin cancers, dermatological conditions (psoriasis) No skin phototoxicity Can be used topically with blue light for skin disorders (Approved, actinic keratoses)... 

A set of N1-unsubstituted furo[2,3- ]quinolin-2(l/f)-ones including 81a-c, 83, and 84a and 84b were prepared by the route shown in Scheme 11, and their photobiological activities were compared with those of the photoche-motherapeutic drugs 4,6,8,9-tetramethylfuro[2,3-, ]quinolin-2(l//)-one (HFQ) and 8-methoxypsoralen 8-MOP <2002BMC2835>. The anti-proliferative activity of these furoquinolinones was tested upon UVA irradiation in mammalian cells, and almost all were found more active than 8-MOP, and free of any mutagenic activity and skin phototoxicity. 

It is widely accepted that photoinduced lipid peroxidation has detrimental effects on cell membranes and therefore plays an important role in skin phototoxicity. The observed photohemolysis induced by these antimalarials may be an indication of extensive photoperoxidation of the membrane lipids. Photo-oxidation experiments with linoleic acid should be performed supplementary to the photohemolysis studies. 

Finally, any reports of skin phototoxicity for a particular drug should provide a clear warning of potential ocular phototoxicity. Skin phototoxicity is more readily apparent than ocular phototoxicity, although it is induced by compounds with similar chemical features (Oppenlander, 1988). 

ALA induced protoporhyrin IX produces excellent fluorescence for diagnostic purposes and is activated at 635 nm. Sufficient cell kill of superficial tumors is achieved at energies of up to 100 J cm 2. The ratio of ALA concentration in tumor to normal brain is around 4 1 however, there are no clinical data for treatment [32,40,42,47]. Skin phototoxicity of ALA is only few hours after application. 

One of the major side effects of these first generation photosensitizers is prolonged skin photosensitivity, requiring sun and light protection for 1-2 months. Another interesting approach utilises antibody-targeted photodynamic therapy. The use of zinc phthalocyanine antibody-MCA complex improved tumor selectivity, avoided skin phototoxicity and resulted in partial remission of the chest wall metas-tases [17]. 

In spite of these promising results, there are some problems still to be solved in current PDT. The major limitation is the nonselective accumulation of the photosensitizers, which could cause severe damage to normal tissue after laser irradiation. Moreover, patients should stay in the absence of sunlight for a long time after the administration of the photosensitizer, because photosensitizers may result in skin phototoxicity. Various delivery methods such as dendrimers, liposomes, micelles, and nanoparticles have been developed to overcome these problems [74, 75]. 

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