Photodynamic therapy with 5-aminolaevulinic acid-induced porphyrins of an amelanotic melanoma in vivo

Abstract

Of particular interest for photodynamic therapy (PDT) are the endogenously formed and photodynamically active porphyrins produced following topical or systemic application of 5-aminolaevulinic acid (ALA), a haem precursor. Having determined the pharmacokinetics and wavelength dependence of PDT with ALA-induced porphyrins, we analysed the porphyrin metabolites in tumour and surrounding skin. The therapeutic efficacy of PDT using ALA-induced porphyrins was investigated. Amelanotic melanomas (A-Mel-3) were implanted subcutaneously in the back of Syrian golden hamsters (body weight (b.w.), 70–80g). After 5–7 days, tumours with a volume of approximately 150 mm 3 were used for PDT ( n = 36). ALA (500 mg kg −1 b.w., pH 6.5) was injected intravenously 45, 90, 150 and 300 min before light irradiation (635 nm, 100 mW cm −2, 100 J cm −2). Tumours with light irradiation only served as controls. The tumour volume was measured after PDT for 28 days. The total porphyrin content was determined in the tumours, the surrounding skin and erythrocytes prior to and 45, 90, 180, 240, 300 and 480 min and 24 h following intravenous injection of ALA (500 mg kg −1 b.w.; n = 32). Porphyrin metabolites were separated by high pressure liquid chromatography (HPLC). Tumour growth was significantly delayed when PDT with ALA was performed 45, 90 or 150 min following intravenous administration. At that time, protoporphyrin (1.8±0.4 nmol g −1), coproporphyrin (2.2±0.5 nmol g −1) and uroporphyrin (1.7±1.4 nmol −1) were the main metabolites in the tumour tissue. Erythrocytes also contained significant amounts of porphyrins (11.8±1.3 nmol g −1). The tumour and surrounding skin exhibited a different pattern of porphyrin metabolites. Unexpectedly, a single treatment of PDT with ALA-induced porphyrins resulted in only one complete remission out of six amelanotic melanomas when the final therapeutic outcome was assessed after 28 days. The therapeutic efficacy of PDT with ALA-induced porphyrins can be positively correlated with the fluorescence kinetics previously determined. The analysis of the porphyrin metabolites in amelanotic melanoma by HPLC indicates that the porphyrin accumulation is not due to a decreased activity of ferrochelatase. Moreover, the photodynamic effects may not be mediated solely by porphyrins localized in the tumour parenchyma, but also by significant amounts of porphyrins in the microvasculature. PDT with this endogenous photosensitizer failed to induce complete remission of the treated tumours despite irradiation at the time of maximum porphyrin concentration using the optimum therapeutic wavelength. Thus PDT with ALA-induced porphyrins is less effective in our model relative to that observed for the exogenous photosensitizer Photofrin® or synthetic porphycenes after a single treatment.