Nele Evens

Synthesis and biological evaluation of carbon-11- and fluorine-18-labeled 2-oxoquinoline derivatives for type 2 cannabinoid receptor positron emission tomography imaging ☆

INTRODUCTION The type 2 cannabinoid (CB(2)) receptor is part of the endocannabinoid system and has been suggested as a mediator of several central and peripheral inflammatory processes. Imaging of the CB(2) receptor has been unsuccessful so far. We synthesized and evaluated a carbon-11- and a fluorine-18-labeled 2-oxoquinoline derivative as new PET tracers with high specificity and affinity for the CB(2) receptor. METHODS Two 2-oxoquinoline derivatives were synthesized and radiolabeled with either carbon-11 or fluorine-18. Their affinity and selectivity for the human CB(2) receptor were determined. Biological evaluation was done by biodistribution, radiometabolite and autoradiography studies in mice. RESULTS In vitro studies showed that both compounds are high affinity CB(2)-specific inverse agonists. Biodistribution study of the tracers in mice showed a high in vivo initial brain uptake and fast brain washout, in accordance with the low CB(2) receptor expression levels in normal brain. A persistently high in vivo binding to the spleen was observed, which was inhibited by pretreatment with two structurally unrelated CB(2) selective inverse agonists. In vitro autoradiography studies with the radioligands confirmed CB(2)-specific binding to the mouse spleen. CONCLUSION We synthesized two novel CB(2) receptor PET tracers that show high affinity/selectivity for CB(2) receptors. Both tracers show favourable characteristics as radioligands for central and peripheral in vivo visualization of the CB(2) receptor and are promising candidates for primate and human CB(2) PET imaging.

Synthesis, in vitro and in vivo evaluation of fluorine-18 labelled FE-GW405833 as a PET tracer for type 2 cannabinoid receptor imaging.

The type 2 cannabinoid receptor (CB₂R) is part of the endocannabinoid system and is expressed in tissues related to the immune system. As the CB₂R has a very low brain expression in non-pathological conditions, but is upregulated in activated microglia, it is an interesting target for visualization of neuroinflammation using positron emission tomography with a suitable radiolabeled CB₂R ligand. In this study, we radiolabelled a fluoroethyl derivative of GW405833, a well known CB₂R partial agonist, with fluorine-18 (half-life 109.8 min) by alkylation of the phenol precursor with 1-bromo-2-[¹⁸F]fluoroethane. In vitro studies showed that FE-GW405833 behaved as a selective high affinity (27 nM) inverse agonist for hCB₂R. [¹⁸F]FE-GW405833 showed moderate initial brain uptake in mice and rats, but a slow washout from brain and plasma due to retention of a radiometabolite. Specific binding of the tracer to human CB₂R was demonstrated in vivo in a rat model with local CB₂R overexpression in the brain. Optimized derivatives of GW405833 that are less susceptible to metabolism will need to be developed in order to provide a useful tracer for CB₂R quantification with PET.

Preclinical evaluation of [11C]NE40, a type 2 cannabinoid receptor PET tracer

INTRODUCTION Up-regulation of the type 2 cannabinoid receptor (CB(2)R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [(11)C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB(2)R. METHODS The selectivity of NE40 for CB(2)R and its toxicity and mutagenicity were determined. [(11)C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [(11)C]NE40 to human CB(2)R (hCB(2)R) was studied in a rat model with hCB(2)R overexpression. RESULTS [(11)C]NE40 shows specific CB(2)R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [(11)C]NE40 showed specific and reversible binding to hCB(2)R in vivo in a rat model with local hCB(2)R overexpression. CONCLUSIONS [(11)C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB(2)R and is a promising candidate for hCB(2)R PET imaging.

Labelling and biological evaluation of [11C]methoxy-Sch225336: a radioligand for the cannabinoid-type 2 receptor

INTRODUCTION The cannabinoid type 2 receptor (CB(2) receptor) is part of the endocannabinoid system and has been suggested as mediator of a number of central and peripheral inflammatory processes. In the present study, we have synthesized N-[(1s)-1-[4-[[4-methoxy-2-[(4-[(11)C]methoxyphenyl)sulfonyl)-phenyl]sulfonyl] phenyl]ethyl]methanesulfonamide ([(11)C]methoxy-Sch225336) and evaluated this new tracer agent as a potential positron emission tomography radioligand for the in vivo visualization of CB(2) receptors. METHODS Sch225336 was demethylated and the resulting phenol precursor was radiolabelled with a carbon-11 methyl group by methylation using [(11)C]methyl iodide, followed by purification by high-performance liquid chromatography. The log P of [(11)C]methoxy-Sch225336 and its biodistribution in normal mice were determined. Enhancement of brain uptake by inhibition of blood-brain barrier (BBB) efflux transporters was studied. Mouse plasma was analysed to quantify the formation of radiometabolites. The affinity of Sch225336 for the human cannabinoid type 1 and type 2 receptor was determined. RESULTS [(11)C]methoxy-Sch225336 was obtained with a decay corrected radiochemical yield of about 30% and a specific activity of 88.8 GBq/mumol (end of synthesis). After intravenous injection in mice, the compound is rapidly cleared from the blood through the hepatobiliary pathway and does not show particular retention in any of the major organs. Polar metabolites were found in mouse plasma. Brain uptake was low despite the favourable log P value of 2.15, which is partly due to efflux by BBB pumps. CONCLUSION [(11)C]methoxy-Sch225336 is a good candidate for in vivo imaging of the CB(2) receptor, although the low blood-brain barrier penetration limits its potential for central nervous system imaging.

A PET Brain Reporter Gene System Based on Type 2 Cannabinoid Receptors

PET of gene expression in the brain may greatly facilitate neuroscience research and potential clinical implementation of gene or cell therapy of central nervous system diseases. To date, no adequate PET reporter system is available for the central nervous system because available tracers either do not cross the intact blood–brain barrier or have high background signals. Here we report the first, to our knowledge, PET reporter system for imaging gene expression in the intact brain. Methods: We selected the human type 2 cannabinoid receptor (hCB2) as a reporter because of its low basal expression in the brain. An inactive mutant (D80N) was chosen so as not to interfere with signal transduction. As a reporter probe we used the 11C-labeled CB2 ligand, 11C-GW405833, which readily crosses the blood–brain barrier. Dual-modality imaging lentiviral and adeno-associated viral vectors encoding both hCB2(D80N) and firefly luciferase or enhanced green fluorescent protein were engineered and validated in cell culture. Next, hCB2(D80N) was locoregionally overexpressed in rat striatum by stereotactic injection of lentiviral and adeno-associated viral vectors. Results: Kinetic PET revealed specific and reversible CB2 binding of 11C-GW405833 in the transduced rat striatum. hCB2 and firefly luciferase expression was followed until 9 mo and showed similar kinetics. Both hCB2 expression and enhanced green fluorescent protein expression were confirmed by immunohistochemistry. Conclusion: Dual-modality imaging viral vectors encoding hCB2(D80N) were engineered, and the reporter system was validated in different animal species. The results support the potential future clinical use of CB2 as a PET reporter in the intact brain.

Non-invasive imaging of the type 2 cannabinoid receptor, focus on positron emission tomography.

The type 2 cannabinoid receptor (CB(2)R) is a relatively new target for drug development, as the receptor was only discovered in 1993. The CB(2)R is mainly expressed in tissues and organs related to the immune system. However, in pathological conditions, mostly inflammatory, a strong upregulation has been observed. Because of its expression in activated microglia, the type 2 cannabinoid receptor might play an important role in pathologies with a neuroinflammatory component. Positron emission tomography provides a sensitive non-invasive imaging technique to study and quantify receptor expression. In this review, the importance of CB(2)R imaging, the current status and the future possibilities for the development of CB(2)R PET radioligands are discussed.