In vitro and in vivo evaluation of Radachlorin® sensitizer for photodynamic therapy

Abstract

This paper reports the evaluation of a new photosensitizer, Radachlorin® in comparison with one of its well known components but used solely, Chlorin e6. The photodynamic properties, cell uptake and localisation of the 2 drugs were compared. In vitro studies were conducted on human adenocarcinoma cells (HT-29) and lung carcinoma cell line (A549). Both dyes showed an absorption maximum between 640 and 650 nm, that were enhanced by serum, with a shifted maximum at 661 nm. In vitro, phototoxicities of Radachlorin® and Chlorin e6 were nearly identical for HT29 and A549 cells. However, Radachlorin® reached its optimal LD50 sooner (0.59 μg ml⁻¹ for 3 h incubation followed by 20 J cm⁻² of 664 nm light (0.02 W cm⁻²)) than Chlorin e6 (0.60 μg ml⁻¹ for 4 h incubation). For in vivo studies, Swiss athymic mice were grafted with human lung carcinoma of the line A549 15 days before intravenous photosensitizer injection. Fluorescence was recorded through an optical fibre spectrofluorimeter using the 666 nm peak for detection. Maximum Radachlorin® fluorescence in tumor was observed 2 h after injection (1412 ± 313 AU). Selectivity was expressed by the calculated tumor-to-skin and tumor-to-muscle ratios. Maximum ratios (1.45 ± 0.14 for tumor-to-skin and 1.95 ± 0.29 tumor-to-muscle) were observed 7 h after injection with Radachlorin. Maximal Chlorin e6 fluorescence was observed 1 h (shortest time interval measured) after injection in all organs and highest tumor-to-muscle ratio (2.56 ± 0.97) 8 h after injection. Chlorin e6 fluorescence in skin was always at least equivalent to tumor fluorescence. Complete response of grafted tumor was achieved (no recurrence observed during 15 days) after 20 mg kg⁻¹ IV injection and 200 J cm⁻² irradiation (0.3 W cm⁻²) with both drugs. Optimal delays between injection and light delivery were between 1 and 7 h with Radachlorin® and 3 h for Chlorin e6 but severe adverse effects were noted for both drugs when drug–light intervals were shorter than 3 h. This suggests that clinical use would be easier with Radachlorin® than Chlorin e6.