Zell- und molekularbiologische Untersuchungen eines EPO-mimetischen Peptides während der präklinischen Phase der Arzneimittelentwicklung

In this doctoral thesis BB68 was identified as a potent monomeric EPO-mimetic peptide. One of the main tasks was to characterize the in vitro effects of the monomeric peptide (BB68), of the homodimeric peptide (AGEM400) and of the multimeric form AGEM400-HES. The centre of the analysis was focused on AGEM400-HES where on the average of five peptide dimers are coupled multivalent to hydroxyethylstarch (HES). AGEM400-HES showed strong EPO-mimetic effects for all investigated parameters. The effects of AGEM400-HES on proliferation, apoptosis and differentiation of hematopoietic cells were analysed and compared to the EPO-induced effects. Dependent on the cell line AGEM400-HES induced proliferation, survival and differentiation in a dose-dependent manner and with an efficacy which is comparable to that of EPO. Experiments with primary cells of bone marrow from human and monkey confirmed the results. Further on investigations of the receptor binding showed that AGEM400-HES bound specifically to the EPO-receptor and induced same signaling pathways as EPO. AGEM400-HES caused the phosphorylation of STAT5 (JAK/STAT-signaling pathway) and of ERK1/2 (Ras/Raf-signaling pathway) in hematopoietic cell lines. After the generation of antibodies against EPO, BB68 and AGEM400 it was analysed if there were any crossreactions between the different antibodies and antigens. But no crossreactions could be detected. Both mimetic peptide forms were immunological completely different from EPO. A very sensitive ELISA for detecting AGEM400 and AGEM400-HES in human serum and rat serum respectively was developed. The detectable concentration range supposable may be in a clinical relevant range and it could be used as a verification procedure for further pharmacokinetic and clinical studies. AGEM400 is a potent and a promising candidate for a novel “EPO-therapy”. The main advantages are the possibility of a chemical synthesis, the biodegradable carrier molecule (HES) and the immunological diversity to EPO. The latter is a very interesting point regarding patients who had developed antibodies against the endogenous and the recombinant protein EPO during application of EPO. A mimetic peptide could be an alternative to correct anemia in these patients.