Design and evaluation of novel PET and SPECT tracers for imaging the monoamine system and the P-gp transporter

SPECT and PET are non-invasive imaging techniques in which radiotracers are used to study biochemical and physiological functions in the living brain. In vivo brain mapping with PET or SPECT can be of great importance in drug development as well as in the effective diagnosis, treatment and management of neurological and psychiatric disorders and substance abuse. Therefore, the development of new and more specific and selective radiotracers is of great importance in neuroimaging. In this thesis, new SPECT or PET radioligands directed towards two different brain elements, being the monoamine system and the P-gp transporter, were developed and evaluated in vivo. The first objective of this thesis was the design of radiotracers for the monoamine system more specifically, the norepinephrine transporter, monoamine oxidase and dopamine transporter which are all responsible for neurotransmitter inactivation. The synthesis, radiosynthesis and preliminary evaluation of [123I]-(S,S)-IPBM, an iodinated reboxetine analogue aimed to visualize the norepinephrine transporter is reported. In a following section, the radiolabelling as well as the in vivo characterization of two [11C]-labelled pyrrole-2-carboxamide derivatives are described. In vitro, they were both potent inhibitors of monoamine oxidase-A. The synthesis along with the radiolabelling and in vivo evaluation of [123I]-FMIP as a selective radiotracer for the dopamine transporter is reported. FMIP has nanomolar affinity for the dopamine transporter and better selectivity over the other monoamine transporters compared to the already existing ligands for dopamine transporter imaging with SPECT. In the last part of this thesis, the focus was redirected towards imaging of the P-glycoprotein transporter. The contribution of P-gp to the low brain uptake of [123I]-FMIP along with its potential as P-gp imaging agent are investigated. Finally, the radiolabelling of an in vitro characterized substrate (MC80) of the P-gp pump with 11C and the evaluation of this tracer in vivo for its potential to image P-gp function or expression is described

N-Linked glycosylation and near-infrared spectroscopy in the diagnosis of prostate cancer

Background: Performing a prostate biopsy is the most robust and reliable way to diagnose prostate cancer (PCa), and to determine the disease grading. As little to no biochemical markers for prostate tissue exist, we explored the possibilities of tissue N-glycosylation and near-infrared spectroscopy (NIR) in PCa diagnosis. Methods: Tissue specimens from 100 patients (benign prostate hyperplasia (BPH), n = 50; and PCa, n = 50) were obtained. The fresh-frozen tissue was dispersed and a tissue N-glycosylation profile was determined. Consequently, the formalin-fixed paraffin-embedded slides were analyzed using NIR spectroscopy. A comparison was made between the benign and malignant tissue, and between the various Gleason scores. Results: A difference was observed for the tissue of N-glycosylation between the benign and malignant tissue. These differences were located in the fycosylation ratios and the total amount of bi- and tetra-antennary structures (all p < 0.0001). These differences were also present between various Gleason scores. In addition, the NIR spectra revealed changes between the benign and malignant tissue in several regions. Moreover, spectral ranges of 1055-1065 nm and 1450-1460 nm were significantly different between the Gleason scores (p = 0.0042 and p = 0.0195). Conclusions: We have demonstrated biochemical changes in the N-glycan profile of prostate tissue, which allows for the distinction between malignant and benign tissue, as well as between various Gleason scores. These changes can be correlated to the changes observed in the NIR spectra. This could possibly further improve the histological assessment of PCa diagnosis, although further method validation is needed

Radiosynthesis, in vitro and in vivo evaluation of I-123-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, [I-123]-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 [H-3]-anandamide, demonstrated interaction of 2 and 4 with FAAH (IC50 values of 5.78 lM and 3.14 lM, respectively). [I-123]-2 and [I-123]-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 [I-123]-2 and 0.58% ID/g at 10 min pi for [I-123]-4). However, stability studies demonstrated the sensitivity of both tracers to dehalogenation. (c) 2008 Elsevier Ltd. All rights reserved

Effect of cyclosporin A administration on the biodistribution and multipinhole μSPECT imaging of [123-I]R91150 in rodent brain

Purpose P-glycoprotein (Pgp) is an efflux protein found amongst other locations in the blood–brain barrier. It is important to investigate the effect of Pgp modulation on clinically used brain tracers, because brain uptake of the tracer can be altered by blocking of the Pgp efflux transporter. The function of Pgp can be blocked with cyclosporin A. Methods We investigated the effect of cyclosporin A administration on the biodistribution of [123I]R91150 in rodents, and the effect of Pgp blocking on the quality of multipinhole μSPECT imaging with [123I]R91150. The influence of increasing doses of cyclosporin A on the brain uptake of [123I]R91150 was investigated in NMRI mice. A biodistribution study with [123I]R91150 was performed in male Sprague-Dawley rats pretreated with cyclosporin A and not pretreated. Brain uptake of [123I]R91150 after cyclosporin A injection was compared to the brain uptake in untreated animals, and a displacement study with ketanserin was performed in both groups. A multipinhole μSPECT brain imaging study was also performed using a Milabs U-SPECT-II camera in male Sprague-Dawley rats. To exclude the effect of possible metabolites, a metabolite study was also performed. Results At the highest cyclosporin A dose (50 mg/kg), a sevenfold increase in brain radioactivity concentration was observed in NMRI mice. Also, a dose-response relationship was established between the dose of cyclosporin A and the brain uptake of [123I]R91150 in mice. Compared to the control group, a five-fold increase in [123I]R91150 radioactivity concentration was observed in the brain of Sprague-Dawley rats after cyclosporin A treatment (50 mg/kg). Radioactivity concentration in the frontal cortex increased from 0.24±0.0092 to 1.58±0.097% injected dose per gram of tissue after treatment with cyclosporin A (at the 1-h time-point). Blood radioactivity concentrations did not increase to the same extent. The cortical activity was displaced by administration of ketanserin. A metabolite study confirmed that there was no increased metabolism of [123I]R91150 due to cyclosporin A. The visual quality of multipinhole μSPECT images with [123I]R91150 in Sprague-Dawley rats improved markedly after cyclosporin A pretreatment. Conclusion From the results obtained in the biodistribution studies, it can be concluded that [123I]R91150 is a substrate for Pgp in rodents. A relationship between the administered dose of cyclosporin A and the increase in [123I]R91150 brain radioactivity concentration was established. The overall quality of our multipinhole μSPECT images with [123I]R91150 in rats improved markedly after pretreatment of the animals with cyclosporin A

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, FAAH's 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 C-11 in good yields and high radiochemical purity and displayed brain uptake in C57BL/6J mice. Radioligands [C-11]-17 and [C-11]-18 merit further investigation in vivo

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 [11C]-labelled pyrrole-2-carboxamide derivates, RS 2315 and RS 2360, along with the characterization of their in vivo properties. Methods: The radiolabelling of [11C]-RS 2315 and [11C]-RS 2360 was accomplished by alkylation of their amide precursors with [11C]CH3I. Biodistribution, blocking and metabolite studies of both tracers were performed in NMRI mice. Finally, a PET study in Sprague-Dawley rats was performed for [11C]-RS 2360. Results: Both tracers were obtained in a radiochemical yield of approximately 30% with radiochemical purity of &gt;98%. Biodistribution studies showed high brain uptake followed by rapid brain clearance for both radiotracers. In the brain, [11C]-RS 2360 was more stable than [11C]-RS 2315. Blocking studies in mice could not demonstrate specificity of [11C]-RS 2315 towards MAO-A or MAO-B. The blocking and imaging study with [11C]-RS 2360 on the other hand indicated specific binding in MAO-A at the earliest time points. Conclusions: [11C]-RS 2315 displayed a high nonspecific binding and is therefore not suitable for visualization of MAO-A in vivo. [11C]-RS 2360 on the other hand has potential for mapping MAO-A since specific binding is demonstrated. © 2010

PET imaging of fatty acid amide hydrolase in the brain : synthesis and biological evaluation of an C-11-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 [C-11]-4-methoxyphenylcarbamate or [C-11]-1, as potential FAAH imaging agent. Methods: The inhibitory activity of 1 was determined in vitro using recombinant FAAH. Radiosynthesis of [(CH)-C-11]-1 was performed by methylation using [C-11]-CH3I, 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: [C-11]-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 [C-11]-1. The inhibition mechanism was determined to be reversible. In addition, the inhibition of URB597 appeared slowly reversible. Conclusions: Although [C-11]-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. (C) 2010 Published by Elsevier Inc