The kinetics of the formation of biologically active psoralen photooxidation (POP) products were analyzed by the biological effects produced. Effects of the UV light fluence rate and psoralen concentration during the preirradiation were investigated to assess the yield of POP products, which were active in vivo (inducing suppression of delayed-type hypersensitivity [DTH] reaction to sheep red blood cells) and in vitro (altering the human erythrocyte membrane permeability). It was shown that the reciprocity law of the irradiation fluence rate and time was not valid in the case of POP-induced hemolysis and DTH suppression. Immunosuppressive POP products were more efficiently formed at low fluence rate (20.8 W/m2), whereas POP hemolysins were more efficiently produced at a high fluence rate (180 W/m2) of UV light. The yield of immunosuppressive POP products was enhanced in dilute psoralen solutions, while the POP hemolysins yield increased with increasing psoralen concentration. A kinetic scheme for psoralen photoproduct formation was proposed. Kinetic analysis showed that a labile intermediate was produced as the result of excitation of psoralen. This intermediate was either converted to a stable immunosuppressive POP product, or two intermediates combined to form a POP hemolysin. It is proposed that PUVA therapy conditions are more favorable for the formation of immunosuppressive rather than membrane-damaging psoralen photooxidation products.

It has been proposed that the therapeutic effect of PUVA (psoralens+UVA radiation) is connected to its immunomodulative properties, and that the molecular basis of such properties is the oxygen-independent photoaddition of psoralens to DNA. We have investigated effects of preliminary photooxidized psoralens (POP) on the delayed-type hypersensitivity reaction (DTH) to sheep red blood cells and on growth of grafted T-cell lymphoma EL-4 in mice. We have shown that intravenous injection of POP at low concentrations activated, and at high concentrations suppressed, DTH. The POP products are thermolabile. They preserved their immunosuppressive activity for 3 days at room temperature and lost it in several min at 58 degrees C. Incubation of POP in the presence of Fe2+ during 2 h before intravenous injection leads to complete loss of its immunomodulative activity, suggesting a peroxidic nature of POP products. The POP-inhibited growth of grafted T-cell lymphoma independent of the mode of POP application in mice (intravenous or subcutaneous injections, oral or nasal administration). Our data suggest that photooxidative reactions of psoralens, in addition to oxygen-independent photoaddition to DNA, form the basis for biological activity of these drugs.

The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.

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