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Dr. Thomas Schulze

Dissertation: Entwicklung einer In-Vitro-Methode zur Beurteilung des photosensibilisierenden Potentials von Arzneistoffen

The aim of this work to evaluate the photochemical behavior of drugs and their associated photosensitizing potential by instrumental methods was successfully achieved. In this investigation, an HPLC method was developed, which makes it possible to determinate reactive oxygen species generated from photoirradiated drugs. This was achieved by using various surrogate parameters to detect and create kinetics for the reactive oxygen species. The formation of singlet oxygen under the influence of light was studied with the help of FFA and RNO to various drugs. In the process ketoprofen, furosemide and doxycycline showed the strongest 1O2 formation. Furthermore, it is possible to determine with this method whether the 1O2 is formed directly by the drug or by one of its photoproducts. The formation of superoxide was detected using NBT. The described method allows predicting the photosensitive/phototoxic potential of a drug through a type II reaction. In order to predict the photosensitizing potential of a drug through a type I reaction an electron paramagnetic resonance method was developed. In the developed method, the drug was irradiated during simultaneous spectrum recordings. The result of the drug investigations showed, that it is possible to detect light-induced free radical formation over the time, thus, giving insights into the reactivity and durability of the radicals formed. Moreover, it is possible to determine the position at which the free radical is formed. Accord-ingly, it is possible to distinguish whether a drug reacts phototoxic via a type I or type II reac- tion. In addition to these results, the behavior of irritated drugs in presence of biomolecules has been studied. Calcitonin has been used as a model molecule. It was found that especially the drugs which also showed a pronounced formation of reactive oxygen species, lead to a degra-dation of calcitonin. Not all tested photosensitizers, such as doxycycline, showed a degrada-tion of calcitonin. By combining the photoreactor with an LC-ESI-TOF system, resulting photoproducts were determined. These photoproducts could partially be assigned to the photodegradation products described in the literature. The results promise a further development of the method using an MS-MS detector to have a lot of potential, since both, the detection limit is reduced, and it is also possible to identify the newly formed photoproducts. Therefore, it should be possible to create photo-reaction pathways. The main purpose of the developed method is the screening of the photosensitive and photo-toxic potential of drugs and other substances. Through the use of this screening method, it will be possible to predict the phototoxic or photoallergic potential of pharmaceutical substances in early stage of development. Furthermore, statements about the photochemical mechanism of the drug can be made.