Sylvie De Bruyne

Synthesis, In Vitro and In Vivo Evaluation, and Radiolabeling of Aryl Anandamide Analogues as Candidate Radioligands for In Vivo Imaging of Fatty Acid Amide Hydrolase in the Brain

Fatty acid amide hydrolyase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids in the brain. Imaging FAAH in vivo using PET or SPECT is important to deeper understanding of its role in neuropsychiatric disorders. However, at present, no radioligand is available for mapping the enzyme in vivo. Here, we synthesized 18 aryl analogues of anandamide, FAAHs endogenous substrate, and in vitro evaluated their potential as metabolic trapping tracers. Interaction studies with recombinant FAAH revealed good to very good interaction of the methoxy substituted aryl anandamide analogues 17, 18, 19, and 20 with FAAH and they were identified as competing substrates. Compounds 17 and 18 did not display significant binding to CB1 and CB2 cannabinoid receptors and stand out as potential candidate metabolic trapping tracers. They were successfully labeled with 11C in good yields and high radiochemical purity and displayed brain uptake in C57BL/6J mice. Radioligands [11C]-17 and [11C]-18 merit further investigation in vivo.

PET imaging of fatty acid amide hydrolase in the brain: synthesis and biological evaluation of an 11C-labelled URB597 analogue

INTRODUCTION Fatty acid amide hydrolase (FAAH) is part of the endocannabinoid system (ECS) and has been linked to the aetiology of several neurological and neuropsychiatric disorders. So far no useful PET or SPECT tracer for in vivo visualisation of FAAH has been reported. We synthesized and evaluated a carbon-11-labeled URB597 analogue, biphenyl-3-yl [(11)C]-4-methoxyphenylcarbamate or [(11)C]-1, as potential FAAH imaging agent. METHODS The inhibitory activity of 1 was determined in vitro using recombinant FAAH. Radiosynthesis of [(11)C]-1 was performed by methylation using [(11)C]-CH(3)I, followed by HPLC purification. Biological evaluation was done by biodistribution studies in wild-type and FAAH knock-out mice, and by ex vivo and in vivo metabolite analysis. The influence of URB597 pretreatment on the metabolisation profile was assessed. RESULTS [(11)C]-1 was obtained in good yields and high radiochemical purity. Biodistribution studies revealed high brain uptake in wild-type and FAAH knock-out mice, but no retention of radioactivity could be demonstrated. Metabolite analysis and URB597 pretreatment confirmed the non-FAAH-mediated metabolisation of [(11)C]-1. The inhibition mechanism was determined to be reversible. In addition, the inhibition of URB597 appeared slowly reversible. CONCLUSIONS Although [(11)C]-1 inhibits FAAH in vitro and displays high brain uptake, the inhibition mechanism seems to deviate from the proposed carbamylation mechanism. Consequently, it does not covalently bind to FAAH and will not be useful for mapping the enzyme in vivo. However, it represents a potential starting point for the development of in vivo FAAH imaging tools.

Radiosynthesis, in vitro and in vivo evaluation of 123I-labeled anandamide analogues for mapping brain FAAH.

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids, including anandamide. This paper is the first report of the synthesis, [123I]-labeling and in vitro and in vivo evaluation of anandamide analogues as potential metabolic trapping radioligands for in vivo evaluation of brain FAAH. N-(2-iodoethyl)linoleoylamide (2) and N-(2-iodoethyl)arachidonylamide (4) were synthesized with good yields (75% and 86%, respectively) in a two steps procedure starting from their respective acids. In vitro analyses, performed using recombinant rat FAAH and [3H]-anandamide, demonstrated interaction of 2 and 4 with FAAH (IC50 values of 5.78 microM and 3.14 microM, respectively). [123I]-2 and [123I]-4 were synthesized with radiochemical yields of 21% and 12%, respectively, and radiochemical purities were > 90%. Biodistribution studies in mice demonstrated brain uptake for both tracers (maximum values of 1.23%ID/g at 3 min pi for [123I]-2 and 0.58%ID/g at 10 min pi for [123I]-4). However, stability studies demonstrated the sensitivity of both tracers to dehalogenation.

Radiosynthesis and in vivo evaluation of [11C]-labelled pyrrole-2-carboxamide derivates as novel radioligands for PET imaging of monoamine oxidase A

INTRODUCTION Since MAO-A is an enzyme involved in the metabolism of neurotransmitters, fluctuations in MAO-A functionality are associated with psychiatric and neurological disorders as well as with tobacco addiction and behaviour. This study reports the radiolabelling of two [(11)C]-labelled pyrrole-2-carboxamide derivates, RS 2315 and RS 2360, along with the characterization of their in vivo properties. METHODS The radiolabelling of [(11)C]-RS 2315 and [(11)C]-RS 2360 was accomplished by alkylation of their amide precursors with [(11)C]CH(3)I. Biodistribution, blocking and metabolite studies of both tracers were performed in NMRI mice. Finally, a PET study in Sprague-Dawley rats was performed for [(11)C]-RS 2360. RESULTS Both tracers were obtained in a radiochemical yield of approximately 30% with radiochemical purity of >98%. Biodistribution studies showed high brain uptake followed by rapid brain clearance for both radiotracers. In the brain, [(11)C]-RS 2360 was more stable than [(11)C]-RS 2315. Blocking studies in mice could not demonstrate specificity of [(11)C]-RS 2315 towards MAO-A or MAO-B. The blocking and imaging study with [(11)C]-RS 2360 on the other hand indicated specific binding in MAO-A at the earliest time points. CONCLUSIONS [(11)C]-RS 2315 displayed a high nonspecific binding and is therefore not suitable for visualization of MAO-A in vivo. [(11)C]-RS 2360 on the other hand has potential for mapping MAO-A since specific binding is demonstrated.

In vivo evaluation of [123I]-4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl)piperidine, an iodinated SPECT tracer for imaging the P-gp transporter

INTRODUCTION P-glycoprotein (P-gp) is an energy-dependent transporter that contributes to the efflux of a wide range of xenobiotics at the blood-brain barrier playing a role in drug-resistance or therapy failure. In this study, we evaluated [(123)I]-4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl)piperidine ([(123)I]-FMIP) as a novel single photon emission computed tomography (SPECT) tracer for imaging P-gp at the brain in vivo. METHODS The tissue distribution and brain uptake as well as the metabolic profile of [(123)I]-FMIP in wild-type and mdr1a (-/-) mice after pretreatment with physiological saline or cyclosporin A (CsA) (50 mg/kg) was investigated. The influence of increasing doses CsA on brain uptake of [(123)I]-FMIP was explored. microSPECT images of mice brain after injection of 11.1 MBq [(123)I]-FMIP were obtained for different treatment strategies thereby using the Milabs U-SPECT-II. RESULTS Modulation of P-gp with CsA (50 mg/kg) as well as mdr1a gene depletion resulted in significant increase in cerebral uptake of [(123)I]-FMIP with only minor effect on blood activity. [(123)I]-FMIP is relative stable in vivo with >80% intact [(123)I]-FMIP in brain at 60 min p.i. in the different treatment regiments. A dose-dependent sigmoidal increase in brain uptake of [(123)I]-FMIP with increasing doses of CsA was observed. In vivo region of interest-based SPECT measurements correlated well with the observations of the biodistribution studies. CONCLUSIONS These findings indicate that [(123)I]-FMIP can be applied to assess the efficacy of newly developed P-gp modulators. It is also suggested that [(123)I]-FMIP is a promising SPECT tracer for imaging P-gp at the blood-brain barrier.

Radiosynthesis and in vivo evaluation of [11C]MC80 for P-glycoprotein imaging

P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. The in vitro characterized modulator 6,7-dimethoxy-2-(6-methoxy-naphthalen-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (MC80) of the P-gp pump was labelled with (11)C and evaluated in vivo for its potential to image P-gp function and expression. Radiochemical pure (>98%) [(11)C]MC80 was obtained within 25 min starting from [(11)C]methyl iodide with radiochemical yield of 26%. Biodistribution studies in FVB mice demonstrated a high baseline brain uptake (7.66 + or - 1.38%ID/g at 1 min pi). Cerebral uptake was increased in mdr1a knock-out mice as well as after CsA pretreatment. Pre-administration of an excess of non-radioactive MC80 caused a reduced uptake in several target organs including brain, pancreas and intestines. The results indicate that [(11)C]MC80 kinetics are modulated by P-gp. Reversed phase-HPLC analysis of brain revealed an excellent metabolic profile (>90% intact [(11)C]MC80).

An evaluation of FDG-PET as a predictor of metabolic response to chemotherapy in metastatic colorectal cancer patients (mCRC)

e14057 Background: Limited information is available on the role of FDG-PET in the management of patients with mCRC. Bevacizumab prolongs overall survival and progression-free survival (PFS) when added to standard chemotherapy in mCRC patients. The purpose of the study was to explore the possible role of FDG-PET as response predictor for bevacizumab treatment in liver mCRC patients. METHODS Eighteen mCRC patients (7 women and 11 men), were treated with FOLFOX (n=12) or FOLFIRI (n=6) and bevacizumab (5 mg/kg). FDG-PET scans were performed before treatment (baseline) and median 15 days after cycle 5 (follow-up). PET scans were read by a nuclear medicine physician, blinded to the results of clinical parameters. The most representative liver lesion was used as target lesion. PET results were quantified by calculating the maximum standard uptake value (SUVmax) at baseline and follow-up. PFS was defined as the time from start of treatment to the date of first documented disease progression. RESULTS Median PFS was 10 months (95% CI: 8.51-11.49). The median baseline SUVmax was 5.18 (IQR: 3.61-8.42) and the median follow-up SUVmax was 2.93 (IQR: 2.15-5.94; p=0.025). Following the EORTC criteria, FDG-PET revealed a complete metabolic response in 8 patients (44%), partial metabolic response in 3 patients (17%), stable metabolic disease in 3 patients (17%) and progressive metabolical disease in 4 patients (22%). Correlation between metabolic response and radiological response according to RECIST was as follows: 6 of the 8 patients with radiological response had metabolic response (sensitivity 75%) and 5 out of 10 patients with radiological stable or progressive disease were metabolic non-responders (specificity 50%). Using the Cox proportional hazards model, the follow-up SUVmax (HR 1.33, 95% CI 1.000-1.776, p=0.05) showed a trend towards significant prediction of PFS. The Kaplan-Meier log rank test showed a PFS benefit for patients with >30% reduction in their SUVmax (p=0.02). CONCLUSIONS The use of FDG-PET for therapy monitoring is clinically feasible and the follow-up SUVmax seems to be a promising predictor of PFS.

Preoperative chemotherapy for colorectal liver metastases : prediction of response by DCE-MRI abd FDG-PET/CT?

Human primary immunodeficiencies (PIDs) comprise a broad group of inherited disorders characterised by developmental or functional defects of myeloid or lymphoid haemapoietic-derived cells, as well as non-haemapoietic cells involved in protective immunity. More than 150 different forms of PIDs affecting distinct components of the innate and adaptive immune system, such as neutrophils, macrophages, dendritic cells, complement proteins, natural killer cells, and T and B lymphocytes have been described [1]. Clinically, PIDs can be associated with any combination of infectious disease, autoimmunity, auto-inflammatory, allergy and malignancy. The best known PIDs show Mendelian inheritance and first become symptomatic during childhood. However, the field of PIDs is rapidly expanding and PIDs showing non-Mendelian inheritance and/or affecting primarily adult patients is being increasingly recognized. Conventional PIDs are typically seen as rare monogenic conditions associated with detectable immunologic abnormalities, resulting in a broad susceptibility to multiple and recurrent infections caused by weakly pathogenic and more virulent microorganisms. By opposition to these conventional PIDs, nonconventional primary immunodeficiencies as Mendelian conditions manifesting in otherwise healthy patients as a narrow susceptibility to infections, recurrent or otherwise, caused by weakly pathogenic or more virulent microbes are now reported [2]. By now, up to 120 diseasecausing genes have been identified. This molecular characterisation of PIDs has helped to increase our understanding of their physiopathology. The study of these diseases has provided essential insights into the functioning of the immune system with the ultimate goal of facilitating diagnosis and treatment.