Development of continuous beds and coated open tubular columns for chiral capillary electrochromatography and microbore HPLC.

Research project P 13815 New chiral phases for chiral capillary electrochromatography Martin SCHMID 28.06.1999 Objective of this project is the synthesis of new types of chiral phases for microbore (capillary) high performance liquid chromatography (microbore HPLC) and capillary electrochromatography (CEC) and their application to the chiral separation of compounds of biological and pharmacological interest. The development of methods for chiral separations has become very important in recent years since it became evident that the biological or pharmacological activity of chiral compounds is mostly restricted to one of the enantiomers. The "inactive" enantiomer can exhibit unwanted side effects, in some cases antagonistic effects or even toxic effects. To avoid the expensive preparation of phases on silica basis and complicated packing of the capillaries connected with the need of circumstantial preparation of frits by sintering the silica gel packing, innovative approaches will be developed for the preparation of capillaries with chiral phases. Two new technologies will be followed: The synthesis of polymeric continuous beds containing the chiral selector incorporated by in-situ polymerization in the capillary and the preparation of open tubular capillaries coated with chiral polymer nanoparticles. In addition to advantages with respect of avoiding packing problems, such polymer phases described would permit higher flow rates, resulting in lower separation times. Furthermore, these capillaries would be significantly less expensive. The capillaries prepared will be applied to the chiral separation of sympathomimetics, betha-blockers, antiinflammatory drugs of the profen type and various other drug classes using capillary HPLC and CEC.

Adenocarcinoma of the lung has developed into the most common lung tumor in industrialized countries. Atypical adenomatous hyperplasia is so far the only known precursor lesion of adenocarcinomas. It can be divided in a low and a high grade variant. By genetic analysis we have shown that high grade AAH shows a great overlap of chromosomal aberrations with the bronchioloalveolar carcinoma and for that reason we have proposed, that high grade AAH should not be interpreted as preneoplasia, but as early bronchioloalveolar adenocarcinoma of the lung, irrespective from its size. Further we have described a new precursor lesion we have termed bronchiolar columnar cell dysplasia (BCCD). By means of lasermicrodissection and molecular-cytogenetic techniques we have demonstrated that even small clusters of these dysplastic cells, not exceeding 1mm in diameter, are affected by extensive chromosomal gains or losses. A third precursor lesion has been identified in childhood CCAM, and proven to be a forerunner for childhood adenocarcinomas of the lung. In addition these adenocarcinomas are distinct from adenocarcinomas in adults, either smokers or non-smokers. The development of a tumor is, at least at the beginning, strongly connected to its environment. In literature the question has been raised, if changes in the tumor associated stroma can promote or initiate tumor development. To address this question we have analyzed adenocarcinomas and squamous cell carcinomas of the lung and their intermingled stroma for chromosomal aberrations. In contrast to other organs, changes within the stroma do not seem to play that important role. However, by the detection of stress hormones and enzymes involved in the detoxification of carcinogenic substances, it was possible to demonstrate that parenchyma nearby the tumor is exposed to stress and toxic substances, while parenchyma far away is not. Whereas normal stroma cells are shielded by the epithelium, this effect is lost in the tumor-stroma areas. Changes in the DNA of a cell are the first step in tumorigenesis, however, not all genes located at the imbalanced chromosomal region have an impact on tumor development. In order to figure out the key players we have tracked down the changes in DNA to the RNA and protein level. At both levels we utilized recently developed array techniques. RNA was investigated by hybridizing it on small arrays of thousands of DNA spots mounted on glass slides representing single genes. Proteins were analyzed by immunohistochemistry on small tissue cores arrayed in a recipient paraffin block. At the end we came up with several proteins that have turned out useful as prognostic markers and targets for new therapeutic approaches.