Synthesis, evaluation and structural analysis of new ligands for the integrin alpha v beta 6 for PET imaging of liver fibrosis
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Abstract
Liver fibrosis is a very frequent disease and often leads to the necessity of transplantation for the patient. This is due to the lack of any anti-fibrotic drugs – therefore, the typical treatment is purely causative. But the treatment of an underlying hepatitis B/C, non-alcoholic-steato hepatitis or alcohol abuse are not always sufficient in order to halt the diseases progression. Furthermore, the current diagnostic ‘gold standard’ of a biopsy is not ideal – the sample represents only a 1/50.000 of the whole organ, which can cause false-positive as well as false-negative results. Especially for intermediate stages, it is difficult to obtain information allowing conclusions about treatment efficiency. For that reason, the positron emission tomography (PET) is thought to be a promising technique, which could overcome the current diagnostic pitfalls, if a suitable tracer substance was given.
The development of such a substance was the aim of this work. The integrin αvβ6 was chosen as the target structure, as it is directly involved in the fibrogenetic pathway and furthermore in the liver it is solely expressed by cholangiocytes. The high affinity ligand EMD527040 was therefore synthesized and two derivatives thereof were produced – to be used as labelling precursors for 68Ga, an easily available PET nuclide. For both substances, convenient labelling procedures were developed, yielding radiochemical yields of >95 %.
Stability studies over two hours as well as the determination of their lipophilicity have successfully be performed. The in vitro studies on αvβ6 bearing cells though, did not show any retention on the receptor. Also tests on the isolated and immobilized integrin could not show a binding. This was supported from the biodistribution studies and PET images of both tracer substances – no significant difference between healthy and fibrotic mice was observed. Nevertheless, the more lipophilic substance was apparently metabolized via hepato biliary pathways while the more hydrophilic one was cleared via the kidneys. The logD value for further derivatives should therefore be below -0.19.
As these results were unexpected, a computational study was initiated – a homology model of αvβ6 was derived from a template crystal structure of αvβ3. Furthermore, the electronic as well as electrostatic characteristics of literature known ligands (partially derivatized at the same position) were investigated and also docked to the homology model. A hypothesis could be derived which explains why our ligands did unexpectedly not bind to the target integrin and allowed the proposal of more promising structures.