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Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

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Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesiveness, an important parameter in cancer metastasis. These alterations result from cell sensitivity to photosensitizers and the distribution of photosensitizers in cells. The efficacy of photosensitizers depends on their close proximity to targets and thus on their pharmacokinetics at the cellular level. We studied the cellular distribution of photosensitizers with a confocal microspectrofluorimeter by analysing the fluorescence emitted by benzoporphyrin derivative-monoacid ring A (BPD-MA) and Photofrin relative to their cell sensitivity. Two cancer cell lines of colonic origin, but with different metastatic properties, were used: PROb (progressive) and REGb (regressive). For BPD-MA (1.75 microg/ml), maximal fluorescence intensity (8,300 cts) was reached after 2 h for PROb and after 1 h (4,900 cts) for REGb. For Photofrin (10 microg/ml), maximal fluorescence intensity (467 cts) was reached after 5 h for PROb and after 3 h (404 cts) for REGb. Intracellular studies revealed stronger cytoplasmic than nuclear fluorescence for both BPD and Photofrin. Both of the sensitizers induced a dose-dependent phototoxicity; LD50 with BPD-MA was 93.3 ng/ml for PROb and 71.1 ng/ml for REGb, under an irradiation of 10 J/cm2. With Photofrin, LD50 was 1,270 ng/ml for PROb and 1,200 ng/ml for REGb under an irradiation of 25 J/cm2. The photosensitizer effect within PROb and REGb cancer cells was assessed by incorporation kinetics and toxicity-phototoxicity tests. The intracellular concentration of the photosensitive agent was one important factor in the effectiveness of PDT, but not the only one contributing to the photodynamic effect. In conclusion, this study showed that there was a clear difference between sensitizer uptake and phototoxicity, even in cancer cells of the same origin. This could induce cell-killing heterogeneity in clinics.

Glucose and amiloride did not change PpIX fluorescence in HT 29 tumor after intraperitoneal injection of aminolevulinic acid but enhanced aminolevulinic acid-photodynamic therapy efficacy. This was probably a result of mechanisms other than an increase in aminolevulinic acid cellular penetration and PpIX production, such as susceptibility to free radical toxicity or alteration of cellular repair enzymes under acidotic conditions. If a decrease of pH induces a more efficient photodynamic therapy as suggested by our results, an easier way to obtain this decrease than glucose and amiloride would be necessary for clinical applications.

New sulfonamide and sulfonic ester porphyrins were prepared and their in vitro characteristics as sensitizers for photodynamic therapy were assessed. Fluorescence spectra were characterised by two main peaks, located between 650-680 nm and 705-740 nm, respectively. Some of these new porphyrins (three out of nine) showed good photodynamic properties in the in vitro assays. In the absence of light, these porphyrins are not toxic. With 50 J.cm−2 illumination light (514 nm) they induced mortality in 50% of HT29 cells with 2 to 4.5 μg/ml. Comparison of in vitro phototoxic efficacy of these three compounds with other photosensitizers already described confirms interest in their phototoxic properties.