Evaluation of [18F]FE@SNAP and [11C]SNAP-7941 as Ligands for the MCH1 Receptor

Background: Changes in the expression of the Melanin concentrating Receptor 1 (MCHR1) have been shown to be involved in a variety of pathologies, especially diabetes, obesity and dysregulation of metabolic feedback Mechanisms and depression and anxiety disorders. Preliminary evaluations of SNAP-7941 on MCHR1 showed high affinity and selectivity. Aim of the study: The present study aims at the evaluation of the new radioligands [ 18F]FE@SNAP and [ 11C]SNAP-7941, targeting the MCHR1. SNAP-7941, the mother compound which is chemically closely related, has been described to display a good affinity (Kd 0.18nM) for the MCHR1 and on the basis of the existing literature it is conclusive, that the MCHR1 can serve as a new promising target for the PET-imaging of various pathologies. Materials and Methods: The preparation of [ 18F]FE@SNAP will follow the standard procedure established at our department using distilled 1-bromo-2-[ 18F]fluoroethane ([18F]BFE). Precursor ("SNAP-acid") will be added to the distilled [ 18F]BFE. Purification will be done by semi-preparative HPLC techniques and/or solid phase extraction methods. The preparation of [ 11C]SNAP-7941 will follow standard methylation procedures. To the same precursor [ 11C]methyl iodide will be added and the resulting solution will be purified similarily. Male rats will be injected with [ 18F]FE@SNAP and sacrificed at various time points. Organs will be removed, weighed and counted. CHO cells stably transfected with recombinant human MCHR1 will be cultured after standard protocols. Cells will be transferred to micronic tubes, centrifuged and resuspended. The cell suspension will be thawed and homogenized by sonication. The membrane pellet will be resuspended and protein concentration will be determined. Binding assays will be performed in the presence of the test compounds and quantified by gamma-counting. For ex vivo autoradiography, [ 18F]FE@SNAP will be administered via the tail vein and brain slices will be prepared and subjected to autoradiography. Micro PET experiments in rats will be performed to evaluate biodistribution, kinetics and specific binding of [ 18F]FE@SNAP in a preclinical PET system. Selectivity will be tested against various receptor and transporter systems. For determination of metabolic stability, substrate will be incubated with carboxylesterase. Determination of Michaelis-Menten kinetic parameters will be performed with increasing substrate concentrations and constant enzyme concentration. Main outcome parameters: (1) successful and reproducible preparation of [ 18F]FE@SNAP and [ 11C]SNAP-7941 (2) preclinical evaluation of [ 18F]FE@SNAP and [ 11C]SNAP-7941: Affinity and lipophilicity; Metabolic stability; Biodistribution experiments; Autoradiography ex-vivo and in-vitro and -PET experiments (3) quantitative and qualitative evaluation of the MCHR1 as an appropriate target for PET On the basis of these three evaluations the main aim will be the decision upon a further clinical application of this new MCHR1 concept.

The melanin concentrating hormone receptor 1 (MCHR1) is discussed to be involved in a variety of pathologies, such as obesity, diabetes, depression or gut inflammation all lifestyle diseases of our days. Therefore, MCHR1 has become a very interesting pharmacological target since its discovery in 1999. MCHR1 is predominantly expressed in the brain; besides, it is also found in peripheral tissues, such as adipocytes, colonic epithelial cells and beta- cells of the pancreas. Several MCHR1 antagonists were presented in the last decade; some of them have entered clinical trials for the treatment of obesity, while some are in discussion of becoming anti- diabetic drugs. However, to enable confidence in preclinical to clinical translation of MCHR1 pharmacology, a suitable positron emission tomography (PET) tracer needs to be developed. PET is a non-invasive technique, which permits in vivo visualization and quantification of receptors (amongst others) at a molecular level. On the basis of the highly potent MCHR1 antagonist SNAP-7941, two PET-tracers were developed and evaluated in this thesis. [11C]SNAP-7941, the radioactive counterpart of SNAP7941, was developed first. The second tracer was the [18F]fluoroethylated analogue [18F]FE@SNAP, which has the advantage of the longer half-life of fluorine-18 compared to carbon-11. For both tracers, a suitable radiosynthesis method had to be developed. [11C]SNAP-7941 could be synthesized via [11C]methyl trifalte starting from the precursor SNAP-acid. The synthesis of [18F]FE@SNAP was only possible via a microfluidic device by direct [F]fluorination of a tosylated precursor (Tos@SNAP). Both tracers could be synthesized in a reliable and feasible manner. Preclinical in vitro evaluation included binding affinity studies on CHO-cell expressing the human MCHR1, plasma stability testing and determination of the plasma free fraction, stability testing against liver microsomes and carboxylesterases, determination of the lipohilicity via logD and immobilized artificial membrane (IAM) chromatography and autoradiography on rat brain and human brain tissues. Additionally, [11C]SNAP-7941 was also preclinically evaluated in vivo. Biodistribution and small animal PET experiments with rats were conducted.