Evaluation of (18F)FE@SUPPY as a Ligand for the A3 Receptor

Background and Rationale: Changes of the adenosine A3 receptor subtype (A3AR) expression have been shown in a variety of pathologies, especially (1) neurological and affective disorders, (2) cardiac diseases, (3) oncological diseases and (4) inflammation processes. But the published data, so far, mainly base on ex vivo or post mortem studies. On the basis of these preliminary results in the evaluation of the involvement of the A3AR in these pathologies, the overhead rationale for the present study is the verification of these assumptions with the high end technique of PET. Preliminary evaluations of FE@SUPPY on adenosine receptors showed high affinity and selectivity for the A3AR. Prior to the potential application of [ 18F]FE@SUPPY in clinical studies, various preclinical evaluations (such as biodistribution, micro PET experiments and metabolic tracer stability testing) have to be performed to prove tracer safety. On the other hand, comprehensive quantitative data on the A3AR are still missing and therefore our aim is to evaluate whether the A3AR represents a suitable target for PET imaging using state of the art preclinical experimental set-ups. Aim of the study: The present study aims at the evaluation of the new radioligand [ 18F]FE@SUPPY, targeting the A3AR. FE@SUPPY has been described to display a good affinity (Ki 4.22 nM) for the A3AR and on the basis of the existing literature it is conclusive, that the A3AR can serve as a new promising target for the PET-imaging of various pathologies. Materials and Methods: Chemistry The tosylate derivative will be realized by a new synthetic route, exploiting a highly efficient Me(II) mediated transesterification method. [ 18F]FE@SUPPY will be produced in a one-step one-pot procedure: The activated, dried [ 18F]fluoride-aminopolyether complex will be converted to [ 18F]FE@SUPPY by addition of Tos@SUPPY in acetonitrile at elevated temperature. Biodistribution experiments: male rats will be injected with [ 18F]FE@SUPPY and sacrificed at various timepoints. Organs will be removed, weighed and counted. Radioactivity will be expressed as percentage injected dose per gram tissue. Autoradiography: For ex vivo autoradiography, [ 18F]FE@SUPPY will be administered via the tail vein and brain slices of 20-50 m will be prepared and subjected to imaging. Baseline experiments will be conducted to establish optimal times and incubation temperatures. And for specificity experiments brain slices will be pre-incubated with several blocking agents. Micro PET experiments in rats will be performed to evaluate biodistribution, kinetics and specific binding of [ 18F]FE@SUPPY in a preclinical PET system using different models. Additionally, the selectivity will be tested against various receptor and transporter systems. Metabolic stability testing: Microsomes will be prepared from rat livers, incubations will be performed at 37C for periods and analysed by HPLC. For carboxylesterase studies substrate will be incubated with the enzyme at 37C. Determination of Michaelis-Menten kinetic parameters will be performed with various incubations with increasing substrate concentrations and constant enzyme concentration of 2.5 I.U. A linear regression analysis of the concentration versus time plot yields a steady state velocity represented by the slope k. Main outcome variable: Main parameters of the present study will be (1) the successful and reproducible preparation of [ 18F]FE@SUPPY as a new PET tracer (2) the preclinical evaluation of [ 18F]FE@SUPPY: (a) Metabolic stability against carboxylesterases and hepatic enzymes (b) Biodistribution experiments (c) Autoradiography ex-vivo and in-vitro (d) Micro-PET experiments (3) the quantitative and qualitative evaluation of the A3AR as an appropriate target for the successful imaging of pathologies associated with changes in A3AR distribution. On the basis of these three evaluations the main outcome variable will be the decision upon a further clinical application of this A3AR concept.

Background and Rationale: Changes of the adenosine A3 receptor subtype (A3AR) expression have been shown in a variety of pathologies, especially 1. neurological and affective disorders, 2. cardiac diseases, 3. oncological diseases and 4. inflammation processes. But the published data, so far, mainly base on ex vivo or post mortem studies. On the basis of these preliminary results in the evaluation of the involvement of the A3AR in these pathologies, the overhead rationale for the present study is the verification of these assumptions with the high end technique of PET. Preliminary evaluations of FE@SUPPY on adenosine receptors showed high affinity and selectivity for the A3AR. Prior to the potential application of [ 18F]FE@SUPPY in clinical studies, various preclinical evaluations (such as biodistribution, micro PET experiments and metabolic tracer stability testing) have to be performed to prove tracer safety. On the other hand, comprehensive quantitative data on the A3AR are still missing and therefore our aim is to evaluate whether the A3AR represents a suitable target for PET imaging using state of the art preclinical experimental set-ups. Aim of the study: The present study aims at the evaluation of the new radioligand [ 18F]FE@SUPPY, targeting the A3AR. FE@SUPPY has been described to display a good affinity (Ki 4.22 nM) for the A3AR and on the basis of the existing literature it is conclusive, that the A3AR can serve as a new promising target for the PET-imaging of various pathologies. Materials and Methods: Chemistry The tosylate derivative will be realized by a new synthetic route, exploiting a highly efficient Me(II) mediated transesterification method. [ 18F]FE@SUPPY will be produced in a one-step one-pot procedure: The activated, dried [ 18F]fluoride-aminopolyether complex will be converted to [ 18F]FE@SUPPY by addition of Tos@SUPPY in acetonitrile at elevated temperature. Biodistribution experiments: male rats will be injected with [ 18F]FE@SUPPY and sacrificed at various timepoints. Organs will be removed, weighed and counted. Radioactivity will be expressed as percentage injected dose per gram tissue. Autoradiography: For ex vivo autoradiography, [ 18F]FE@SUPPY will be administered via the tail vein and brain slices of 20-50 m will be prepared and subjected to imaging. Baseline experiments will be conducted to establish optimal times and incubation temperatures. And for specificity experiments brain slices will be pre-incubated with several blocking agents. Micro PET experiments in rats will be performed to evaluate biodistribution, kinetics and specific binding of [ 18F]FE@SUPPY in a preclinical PET system using different models. Additionally, the selectivity will be tested against various receptor and transporter systems. Metabolic stability testing: Microsomes will be prepared from rat livers, incubations will be performed at 37C for periods and analysed by HPLC. For carboxylesterase studies substrate will be incubated with the enzyme at 37C. Determination of Michaelis-Menten kinetic parameters will be performed with various incubations with increasing substrate concentrations and constant enzyme concentration of 2.5 I.U. A linear regression analysis of the concentration versus time plot yields a steady state velocity represented by the slope k. Main outcome variable: Main parameters of the present study will be 1. the successful and reproducible preparation of [ 18F]FE@SUPPY as a new PET tracer 2. the preclinical evaluation of [ 18F]FE@SUPPY: (a) Metabolic stability against carboxylesterases and hepatic enzymes (b) Biodistribution experiments (c) Autoradiography ex-vivo and in-vitro (d) Micro-PET experiments 3. the quantitative and qualitative evaluation of the A3AR as an appropriate target for the successful imaging of pathologies associated with changes in A3AR distribution. On the basis of these three evaluations the main outcome variable will be the decision upon a further clinical application of this A3AR concept.