Development of novel fluorine-18 labeled PET radioligands for monoamine oxidase B (MAO-B)

Monoamine oxidases (MAO-A and MAO-B) are important enzymes regulating the levels of monoaminergic neurotransmitters. Selectiv e and irreversible MAO-B inhibitors such as L -deprenyl and rasagiline are clinically used for the t reatment of psychiatric and neurological disorders. Positron em ission tomography (PET) is a non- invasive imaging technique which has widely been util ized to visualize the localization of MAO-B in monkey and human brains and thereby has b een useful for studying neurodegenerative diseases and epilepsy. This thesi s deals with the synthesis and evaluation of novel fluorine-18 labeled PET radioligan ds for detection of MAO-B activity. The present thesis demonstrates that nine fluorinat ed propargyl amines were synthesized and tested for inhibition of MAO-B. In o rder to label those compounds with fluorine-18 seven chloro-precursors and two sulph amidate-precursors were also synthesized by multi step organic synthesis. Radiola beling of six chloro-precursors with fluorine-18 was accomplished by a one-step nucl eophilic substitution reaction. Radiolabeling of two sulphamidate-precursors with fl uorine-18 was performed in two steps, compromising a nucleophilic substitution foll owed by the removal of the protecting group. The incorporation yield of the fluo rination reactions varied from 40- 70%. The radiochemical purity was >99% and the specif ic radioactivities were in a range of 190-240 GBq/μmol at the time of administrat ion. In vitro MAO inhibition and/or autoradiography (ARG) experimen ts demonstrated a high selectivity for MAO-B over MAO-A for five of the compounds namely [ 18 F]fluorodeprenyl, [ 18 F]fluororasagiline, [ 18 F]fluoro- N ,4-dimethyl- N -(prop-2-ynyl) pentan-2-amine, [ 18 F]fluorodeprenyl-D 2 and [ 18 F]fluororasagiline-D 2 . All five compounds were examined by PET and showed a high initial brain uptake in known MAO-B rich regions in cynomolgus monkey. [ 18 F]Fluorodeprenyl showed a kinetic behavior similar to [ 11 C]deprenyl where its fast irreversible binding to th e enzyme renders the distribution of this radioligand in tissue limited by blood flow rather than the MAO-B enzyme concentration. [ 18 F]Fluororasagiline and [ 18 F]fluoro- N ,4-dimethyl- N -(prop-2-ynyl)pentan-2-amine showed continuous increase of the radioactivity throughout the PET measurement that might be an indic ation of a blood-brain barrier penetrating radiometabolite which might in turn compl icate a reliable quantification. Only [ 18 F]fluorodeprenyl-D 2 and [ 18 F]fluororasagiline-D 2 showed fast wash-out from the brain and less accumulation in cortical and sub- cortical regions. Radiometabolite studies demonstrated that both deuterated analogues were more stable measured in monkey plasma when compared to the non-deuterated a nalogues. These results together suggest that both [ 18 F]fluorodeprenyl-D 2 and [ 18 F]fluororasagiline-D 2 may be improved PET radioligands and potential mole cular imaging biomarker candidates for PET studies in neuroi nflammation and neurodegeneration, accompanied with astrocyte activat ion

The norepinephrine transporter (NET) radioligand (S,S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer’s disease: a post-mortem autoradiographic study.

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimer's disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[F-18]FMeNER-D-2. (S,S)-[F-18]FMeNER-D-2 appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[F-18]FMeNER-D-2 in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[F-18]FMeNER-D-2 as a molecular imaging biomarker in AD. (c) 2010 Elsevier Ltd. All rights reserved

Test-retest reproducibility of [11C]-l-deprenyl-D2 binding to MAO-B in the human brain

Abstract Background [11C]-l-deprenyl-D2 is a positron emission tomography (PET) radioligand for measurement of the monoamine oxidase B (MAO-B) activity in vivo brain. The estimation of the test-retest reproducibility is important for accurate interpretation of PET studies. Results We performed two [11C]-l-deprenyl-D2 scans for six healthy subjects and evaluated the test-retest variability of this radioligand. MAO-B binding was quantified by two tissue compartment model (2TCM) with three rate constants (K 1, k 2, k 3) using metabolite-corrected plasma radioactivity. The λk 3 defined as (K 1/k 2) × k 3 was also calculated. The correlation between MAO-B binding and age, and the effect of partial volume effect correction (PVEc) for the reproducibility were also estimated. %difference of k 3 was 2.6% (medial frontal cortex) to 10.3% (hippocampus), and that of λk 3 was 5.0% (thalamus) to 9.2% (cerebellum). Mean %difference of all regions were 5.3 and 7.0% in k 3 and λk 3, respectively. All regions showed below 10% variabilities except the hippocampus in k 3 (10.3%). Intraclass correlation coefficient (ICC) of k 3 was 0.78 (hippocampus) to 0.98 (medial frontal cortex), and that of λk 3 was 0.78 (hippocampus) to 0.95 (thalamus). Mean ICC were 0.94 and 0.89 in k 3 and λk 3, respectively. The highest positive correlation with age was observed in the hippocampus, as r = 0.75 in k 3 and 0.76 in λk 3. After PVEc, mean %difference were 5.6 and 7.2% in k 3 and λk 3, respectively. Mean ICC were 0.92 and 0.90 for k 3 and λk 3, respectively. These values were almost the same as those before PVEc. Conclusions The present results indicate that k 3 and λk 3 of [11C]-l-deprenyl-D2 are reliable parameters for test-retest reproducibility with healthy subjects both before and after PVEc. The studies with patients of larger sample size are required for further clinical applications

Production of [C-11]Carbon Labelled Flumazenil and L-Deprenyl Using the iMiDEV (TM) Automated Microfluidic Radiosynthesizer

In the last decade, microfluidic techniques have been explored in radiochemistry, and some of them have been implemented in preclinical production. However, these are not suitable and reliable for preparing different types of radiotracers or dose-on-demand production. A fully automated iMiDEV (TM) microfluidic radiosynthesizer has been introduced and this study is aimed at using of the iMiDEV (TM) radiosynthesizer with a microfluidic cassette to produce [C-11]flumazenil and [C-11]L-deprenyl. These two are known PET radioligands for benzodiazepine receptors and monoamine oxidase-B (MAO-B), respectively. Methods were successfully developed to produce [C-11]flumazenil and [C-11]L-deprenyl using [C-11]methyl iodide and [C-11]methyl triflate, respectively. The final products 1644 +/- 504 MBq (n = 7) and 533 +/- 20 MBq (n = 3) of [C-11]flumazenil and [C-11]L-deprenyl were produced with radiochemical purities were over 98% and the molar activity for [C-11]flumazenil and [C-11]L-deprenyl was 1912 +/- 552 GBq/mu mol, and 1463 +/- 439 GBq/mu mol, respectively, at the end of synthesis. All the QC tests complied with the European Pharmacopeia. Different parameters, such as solvents, bases, methylating agents, precursor concentration, and different batches of cassettes, were explored to increase the radiochemical yield. Synthesis methods were developed using 3-5 times less precursor than conventional methods. The fully automated iMiDEV (TM) microfluidic radiosynthesizer was successfully applied to prepare [C-11]flumazenil and [C-11]L-deprenyl

Synthesis, Biodistribution, and Radiation Dosimetry of a Novel mGluR5 Radioligand : F-18-AZD9272

The metabotropic glutamate receptor subtype mGluR5 has been proposed as a potential drug target for CNS disorders such as anxiety, depression, Parkinson's disease, and epilepsy. The AstraZeneca compound AZD9272 has previously been labeled with carbon-11 and used as a PET radioligand for mGluR5 receptor binding. The molecular structure of AZD9272 allows one to label the molecule with fluorine-18 without altering the structure. The aim of this study was to develop a fluorine-18 analogue of AZD9272 and to examine its binding distribution in the nonhuman primate brain in vivo as well as to obtain whole body radiation dosimetry. F-18-AZD9272 was successfully synthesized from a nitro precursor. The radioligand was stable, with a radiochemical purity of >99% at 2 h after formulation in a sterile phosphate buffered solution (pH = 7.4). After injection of F-18-AZD9272 in two cynomolgus monkeys, the maximum whole brain radioactivity concentration was 4.9-6.7% of the injected dose (n = 2) and PET images showed a pattern of regional radioactivity consistent with that previously obtained for C-11-AZD9272. The percentage of parent radioligand in plasma was 59 and 64% (n = 2) at 120 min after injection of F-18-AZD9272, consistent with high metabolic stability. Two whole body PET scans were performed in nonhuman primates for a total of 231 min after injection of F-18-AZD9272. Highest uptakes were seen in liver and small intestine, followed by brain and kidney. The estimated effective dose was around 0.017 mSv/MBq. F-18-AZD9272 shows suitable properties as a PET radioligand for in vivo imaging of binding in the primate brain. F-18-labeled AZD9272 offers advantages over C-11-AZD9272 in terms of higher image resolution, combined with a longer half-life. Moreover, based on the distribution and the estimated radiation burden, imaging of F-18-AZD9272 could be used as an improved tool for quantitative assessment and characterization of AZD9272 binding sites in the human brain by using PET

In vivo evaluation in cynomolgus monkey brain and metabolism of [¹⁸F]fluorodeprenyl: a new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO-B) radioligand, [¹⁸F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹⁸F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO-B radioligand. The highest uptake was observed in the striatum, one of the MAO-B-rich regions, with a peak at approximately 2-3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹⁸F]fluorodeprenyl was largely inhibited by preadministration of the MAO-B inhibitor, L-deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO-B. [¹⁸F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹⁸F]fluorodeprenyl is a potential PET radioligand for visualization of MAO-B activity

Implementation of iMiDEV™, a new fully automated microfluidic platform for radiopharmaceutical production

iMiDEV™ microfluidic system is a new automated tool for a small-scale production of radiopharmaceuticals. This new radiochemistry module utilizes microfluidic cassettes capable of producing diversified radiopharmaceuticals in liquid phase reactions in an automated synthesizer. The user interface is intuitive and designed to give the operator all the information required and to allow driving the synthesis either manually or fully automatically. In this work, we have demonstrated liquid phase reaction and presented the first results of an efficient fully automated [18F]NaF radiosynthesis on the iMiDEV™ platform. Different parameters such as a type of cyclotron targets, initial activity, concentration and volume of the fluoride-18 targetry have been investigated in order to elaborate the optimised radiolabelling of the ligand. Single and double sodium [18F]fluoride synthesis procedures have been successfully developed using two chambers of the cassette. A single-dose of radiotracer was produced in an average radiochemical yield of 87% (decay corrected) within 8 min and quality control tests were performed as per European Pharmacopoeia.Title in Web of Science: Implementation of iMiDEV (TM), a new fully automated microfluidic platform for radiopharmaceutical production</p

Characterization of a Novel M4 PAM PET Radioligand [11C]PF06885190 in Nonhuman Primates (NHP)

Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer’s disease (AD). PET imaging of M4 positive allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand [11C]PF06885190 (b) to evaluate the brain distribution of [11C]PF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of [11C]PF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (VT) of [11C]PF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of [11C]PF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis. [11C]PF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of VT from baseline was around −10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of [11C]PF06885190 was observed, these data suggest that [11C]PF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging