The Adenosine A3 Receptor in Oncology - A completely new Target for PET-Imaging

During a precedent project (FWF Nr P19383, awarded to M. Mitterhauser), [18F]FE@SUPPY, an adenosine A3 receptor (A3R) antagonist was developed and evaluated as the first PET-tracer for the A3R. In general, the A3R is known to be physiologically expressed in human liver, lung, brain, aorta, heart and testis and is involved in a variety of pathologies. The suitability of the A3R as a therapeutic target and as a biological predictive marker bases on two major facts: The over-expression of the A3R in cancer cells beside low expression in normal cells. Furthermore, a direct correlation between A3R tissue expression levels and disease progression was described in breast and colon cancer. Since antagonists of the A3R can be considered as imaging biomarkers for colon-, mamma-, prostate carcinoma and melanoma, a clinical application of potential tracers could be of great benefit. Therefore, and based on the mostly promising findings in the preclinical evaluation process of the precedent project, [18F]FE@SUPPY is a suitable tracer for the continuation of the project and its evaluation for PET-Oncology. Main workpackages: Affinity studies of FE@SUPPY towards the human A1, A2A and A2B receptors will be assessed based on cell harvesting and direct measurements using Ligand Tracer technology. Additionally, tumour cell lines expressing the human A3R will be tested for abundance and extent of A3R and appropriateness for transplantation into immunocompromised mice. Autoradiographic ex-vivo and in-vitro experiments Human tumour tissues with high abundance of A3R will be subjected to competition studies with [18F]FE@SUPPY. The location and distribution of the A3R in these tissue materials will be assessed. Ex-vivo tumour tissues of the xenograft mice will be analysed, both, with a Cyclone Phosphor Imager and a digital autoradiograph. In-vitro stability testing [18F]FE@SUPPY will be incubated using human and mouse liver microsomes and human and mouse plasma; potential metabolites will be analyzed by radio-HPLC to understand in-vivo characteristics in the selected xenograft mouse model. Transplantation of human tumour cell lines in nude mice Mice will be inoculated with tumour cells expressing reasonable amounts of A3R, and subjected to small animal PET. Small animal PET Mice will be injected with radiotracer ([18F]FE@SUPPY or [18F]FDG) and baseline and blocking scans will be performed. Main expected results (1) Selectivity of FE@SUPPY against the human A1, A2A, A2B adenosine receptor subtypes (2) Selection of appropriate tumour cell-lines, expressing the A3R for transplantation (3) Preclinical evaluation of [18F]FE@SUPPY on these A3R-expressing tumour cell lines via (a) binding studies on these cell lines directly and under blocking conditions (b) metabolic experiments (c) small animal PET experiments with transplanted animals (4) Quantitative and qualitative evaluation of the A3R as an appropriate target for the successful imaging of tumour pathologies associated with changes in A3R distribution.

Positron emission tomography (PET) has become a key tool in cancer diagnosis over the last decades as it is a non-invasive imaging technique with unprecedented sensitivity. PET is based on the distribution and accumulation of a radioactively labelled molecule in the living organism and its subsequent detection. Thereby biological functions and the expression of specific target proteins are visualized. The presented project represents the investigation of the fluoroethyl ester [18F]FE@SUPPY for imaging of the adenosine-3-receptor and includes the complete evaluation of this radioligand with respect to binding profile and kinetics, stability and metabolism in different species, biodistribution in mice and rats, and suitability for tumor imaging. In the course of the intensive evaluation of the radioligand [18F]FE@SUPPY on cell systems, several new methods have been established which will improve the in vitro evaluation of new radiopharmaceuticals in early stages of development. This will also allow to save animal experiments in the future, since more precise statements about the suitability of a radioligand for imaging can already be made on cell systems.