The riboflavin-mediated photooxidation of doxorubicin

Purpose: Previously, it was shown that exposing doxorubicin (ADR) to 365 nm light resulted in the loss of its cytotoxic activity as well as its absorb ance at 480 nm, These processes were much enhanced when mediated by ribofla vin. In the present study we investigated the quantitative and qualitative aspects of ribo-flavin-mediated photodegradation of ADR. Methods: ADR solut ions containing variable concentrations of riboflavin and other agents were exposed to 365 nm light for variable time periods and then the absorbance spectrum of ADR was measured by a double beam spectrophotometer. These meas urements were used to calculate the half-time of the ADR degradation proces s. The degraded ADR solutions were analyzed by chromatography and mass spec trometry. Results: Analysis of the riboflavin effect indicated that a maxim al rate of photolytic degradation of ADR was obtained only after most of th e ADR molecules had formed bimolecular complexes with riboflavin. The retar dation of lumichrome formation by ADR and the inhibition of ADR bleaching b y excess of ascorbic acid suggested that ADR was degraded by a photooxidati on process. Similar spectral changes occurred when ADR was exposed to stron g oxidizers such as sodium hypochlorite and dipotassium hexachloroiridate. Cyclic voltammetry revealed that the oxidation-reduction process of ADR was not electrochemically reversible and therefore the oxidation potential cou ld not be determined accurately; however its value should be between 0.23 a nd 0.78 V. Analysis of the photooxidative process revealed that it was not mediated by the formation of singlet oxygen, superoxide anion radicals, hyd rogen peroxide or hydroxyl radicals, and it is suggested that ADR was oxidi zed directly by the excited triplet riboflavin. The mass spectrograms and t he HPLC chromatograms of photooxidized ADR indicate that the central ring o f ADR was opened and that 3-methoxysalicylic acid was produced by this clea vage. Conclusions: The riboflavin-mediated photodegradation of ADR is an ox idative process resulting in the cleavage of the anthraquinone moiety. 3-Me thoxysalicylic acid was identified as one of the resulting fragments. It is possible that some of the large fractions of the ADR metabolites that are nonfluorescent are the result of an in vivo oxidation of ADR and that 3-met hoxysalicylic acid may play a role in the different biological activities o f ADR.