IMPORTANCE OF TYPE-I AND TYPE-II MECHANISMS IN THE PHOTODYNAMIC INACTIVATION OF VIRUSES IN BLOOD WITH ALUMINUM PHTHALOCYANINE DERIVATIVES

The relative importance of type I and type II mechanisms in the photodynamic treatment of red blood cell concentrates (RBCC) to inactivate viruses was studied using aluminum phthalocyanine tetrasulfonate (AlPcS4), visible light and quenching or enhancing agents of reactive forms of oxygen. Treatment of a human RBCC with 10-13 muM AlPcS4 and 25-26 mW/cm2 visible light resulted in the rapid and complete inactivation of added vesicular stomatitis virus (VSV). The addition of mannitol, glycerol, reduced glutathione (GSH), or superoxide dismutase (SOD), known quenching agents of type I mechanisms, had little to no effect on the rate of inactivation of VSV. Significant inhibition of VSV kill was observed on addition of tryptophan or sodium azide, known quenchers of type II mechanisms. Additionally, the rate of VSV kill was enhanced in the presence of D2O. Taken together, these results indicate a predominant role of singlet oxygen in the inactivation of VSV on photodynamic treatment of RBCC. The relative importance of type I and type II mechanisms on cellular toxicity was also evaluated. Little, if any hemoglobin release was observed on treatment of human or rabbit RBCC with 10 muM AlPcS4 and 44 J/cm2 of visible light in the presence or absence of the above mentioned quenchers. The effect of the addition of quenchers on the recovery and circulatory survival of treated, autologous rabbit RBCC, labeled with Cr-51, was also assessed. As compared to a circulatory half-life (T50) of 10-12 days for the untreated controls, RBCC treated with 30 min light exposure and 5 or 10 muM AlPcS4 or 5 muM AlPc had T50 values of 5 days or less. The addition of 1 mM GSH, 1 mM mannitol, or 1 mM tryptophan prior to treatment with 30 min light exposure and 10 muM AlPcS4 resulted in T50 values of 7.9-8.5, 6.5 and 8.5 days, respectively. A similar T50 enhancement was observed on addition of 0.5 mM tryptophan to RBCC prior to treatment with 10 muM AlPc and 30 min light. We conclude that virus kill in the RBCC operates principally through a type II mechanism. whereas both type I and type II mechanisms contribute to red cell cytotoxicity, as measured by circulatory survival in a rabbit model. Consequently, the specificity of virus kill on treatment of a RBCC by photodynamic processes can be enhanced through the addition of quenchers of type I reactions.