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Castro, Mirna (2005): Pharmacokinetic studies on protoporphyrin IX induced by 5-aminolevulinic acid and its esters in a three-dimensional lung tumor mini-organ culture model. Dissertation, LMU München: Medizinische Fakultät



Lung cancer is one of the most common malignancies in the world and remains the leading cause of cancer death among men and women in developed countries, accounting for more deaths than breast, prostate and colorectal cancers combined. The cure for lung cancer is low (<15%) due to the lack of screening methods, the propensity for early spread, and the inability to cure metastatic disease. However, when people are diagnosed with early stage lung cancer, their chances of 5-year survival can be as high as 90%, hence the importance of methods for early diagnopromising technologies. The purpose of this thesis was to establish an in vitro model for lung cancer and to investigate clinically relevant pharmacokinetic parameters for the optimization of 5-aminolevulinic acid (5-ALA) application. The present study suggests that the in vitro three-dimensional mini-organ model consisting of normal human bronchial mucosa co-cultivated with human lung tumor cells (EPLC-32M1) is a good alternative to mono-layer or even spheroid cell culture due to a much closer similarity to the in vivo situation. It largely obviates the necessity for animal experiments to investigate various clinically relevant questions, e.g. drug pharmacokinetics. The three-dimensional mini-organ model has been applied to elucidate the accumulation ofthe fluorescing photosensitizer protoporphyrin IX (PPIX) after delivery of 5-aminolevulinic acid (5-ALA) or some of its esters: • The optimal concentration for maximal achexyl ester. Fluorescence intensity was taken as parameter for the amount of PPIX in the tissue. • The co-culture model was used to study the pharmacokinetics of 5-ALA-induced PPIX fluorescence and its esters in normal epithelium and in tumorous areas. • The tim• Improvement of the three-dimensional mini-organ model by using GFP-transfected EPLC-32M1 lung tumor cells in the organ co-culture system was studied. SUMMARY 62 tumor and her than in lipophilic nature, an enhanced tissue penetration has been an attempt to further enhance the co-culture model, green fluorescent protein (GFP) expression plasmid-transfected lung tumor cells were used in the mini-organ model. Comparable results were obtained in the pharmacokinetics of 5-ALA between co-cultures made with GFP transfected and untransfected lung tumor cells. But GFP expression highly improved the experiment conditions, whereby a higher contrast was reached at the tumor/ normal epithelium boundaries. 5-ALA-induced PPIX fluorescence showed marked differences in the kinetics in normal epithelium as the concentration of PPIX within tumorous areas was hignormal tissue. PPIX fluorescence in tumor increased faster in most of the cases than in normal tissue, but also tended to decay earlier. The results of this study show that the relative fluorescence intensities of PPIX in tumor and normal epithelium are a function of the incubation time, concentration, distribution, and kinetics in tumorous and normal tissue. A number of 5-ALA derivatives are being used in order to modify and improve the tissuedistribution of the PPIX. The tumor/normal epithelium ratios (T/NE ratio) of PPIX fluorescence induced by 5-ALA and its esters were comparable and showed in general a good contrast in the three-dimensional mini-organ model. However, comparing 5-ALA with 5-ALA hexyl ester, 5-ALA hexyl ester induced a 2-fold higher PPIX fluorescence intensity in tumor as well as in normal epithelium at a 5 times lower concentration (0.24 mM). A slight tendency to a rapid PPIX accumulation in tumor and in normal epithelium was observed in co-cultures incubated with 5-ALA methyl ester. In addition, maximal PPIX fluorescence intensities in these co-cultures show individual variations in tumor as well as in normal epithelium. The highest T/NE ratio was observed with 5-ALA butyl ester, but also an inhomogeneous distribution of PPIX in tumorous and normal areas. In general, it was possible to use the same mini-organ model for further experiments, which indicates that 5-ALA esters at their optimal concentrations are not toxic. Amongst the 5-ALA esters, 5-ALA hexyl ester is clearly to be favored as it combines homogeneous distribution, high T/NE ratio, high fluorescence intensities, and the lowest drug concentration needed. Due to itsassumed compared to the other esters as well as 5-ALA. In clinical studies 5-ALA hexyl ester has been used successfully to detect early carcinoma in the urinary bladder by fluorescence imaging. In SUMMARY 63 present work was to establish a model which allows the taken into consideration in the present mini- can be applied to localize One of the main targets of theoptimization of application parameters for 5-ALA-based fluorescence diagnosis for the early detection of lung cancer. The maximum of PPIX fluorescence intensity in the co-culture model was detected between 130 and 180 min after the beginning of incubation with 5-ALA. Clinical results obtained by Huber et al. indicate that a photodynamic diagnosis should be performed approximately 2 hours after topical administration of 5-ALA to patients. Thus the co-culture model corroborates the clinical observation and yields a quantitative confirmation both for the time delay between drug application and fluorescence bronchoscopy (or endobronchial PDT) and for the T/NE contrast that can be expected. The results also suggest that inhalation of 5-ALA hexyl ester at a lower concentration and slightly shorter time interval could enhance the procedures. Since the transport of 5-ALA by blood are notorgan model, the results of this study could primarily be applied to topical application of 5-ALA and its derivatives, in the case of lung cancer per inhalation of these compounds. In order to obtain a significant improvement of patient survival, the number of tumors identified in early stages has to be increased. This could be achieved by enhanced sputum cytology screening. Then, optimized fluorescence bronchoscopyearly lesions with high sensitivity and endoluminal PDT can treat these lesions minimally invasive.