Research ArticleBasic Science Investigations

In Vivo and In Vitro Characterization of a Novel MAO-B Inhibitor Radioligand, 18F-Labeled Deuterated Fluorodeprenyl

Sangram Nag, Patrik Fazio, Lutz Lehmann, Georg Kettschau, Tobias Heinrich, Andrea Thiele, Marie Svedberg, Nahid Amini, Samira Leesch, Ana M. Catafau, Jonas Hannestad, Andrea Varrone and Christer Halldin
Journal of Nuclear Medicine February 2016, 57 (2) 315-320; DOI: https://doi.org/10.2967/jnumed.115.161083

Abstract

The aim of this study was to radiolabel a novel bis-deuterium substituted l-deprenyl analog (fluorodeprenyl-D2) with 18F and to evaluate its potential to visualize and quantify monoamine oxidase (MAO) B activity in vivo. Methods: The precursor compound (5a + 5b) and reference standard (6) were synthesized in multistep syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used to determine inhibitory concentrations of 50%. Radiolabeling was accomplished by a nucleophilic substitution reaction. Whole-hemisphere autoradiography was performed with 18F-fluorodeprenyl-D2. A PET study was performed on a cynomolgus monkey. Radiometabolites were measured in monkey plasma using high-performance liquid chromatography. Results: The 50% inhibitory concentration of compound 6 for MAO-B was 227 ± 36.8 nM. Radiolabeling was accomplished with high radiochemical yield, purity, and specific radioactivity. The autoradiography binding density of 18F-fluorodeprenyl-D2 was consistent with known MAO-B expression in the human brain. In vivo, 18F-fluorodeprenyl-D2 showed favorable kinetic properties, with relatively fast washout from the brain. Regional time–activity curves were better described by the 2-tissue-compartment model. Administration of a 1 mg/kg dose of l-deprenyl yielded 70% inhibition of MAO-B in all regions. Radiometabolite studies demonstrated 20% unchanged radioligand at 120 min after injection. 18F-fluorodeprenyl-D2 showed less irreversibility than did previously reported MAO-B radioligands. Conclusion: The results suggest that 18F-fluorodeprenyl-D2 is a suitable PET radioligand for visualization of MAO-B activity in the human brain.

Monoamine oxidase (MAO) plays an important role in regulating chemical neurotransmitters by catalyzing oxidative deamination of, for example, monoamine neurotransmitters (1). MAO is an important therapeutic target for neurologic disorders such as Alzheimer disease (2), Parkinson disease (3), and depression (4). According to biochemical and pharmacologic properties, this enzyme exists in A and B isoforms (5). In the human brain, MAO-B is the predominant isoform and constitutes up to about 70% of total brain MAO activity (6). MAO-B selectively oxidizes monoamines such as O-tyramine, phenethylamine, and tele-N-methyl histamine and generates hydrogen peroxide, which can react to form highly reactive oxygen species (7). Previously reported increased levels of MAO-B level in Alzheimer disease patients might be associated with a progressive increase in oxidative stress (8) and a consequent reactive astrogliosis (9,10). Because astrocyte activity and, consequently, the activity of the MAO-B system is upregulated in neuroinflammatory processes, radiolabeled MAO-B inhibitors may serve as an imaging biomarker in neuroinflammation and neurodegeneration, including Alzheimer disease (11).

PET, a high-resolution, sensitive, and noninvasive molecular imaging technique, has been successfully used in visualizing the localization of MAO-B activity for studying neurodegenerative diseases (12). Several PET radioligands have been developed to study MAO-B activity, such as 11C-pargyline (13), 11C-deprenyl (12), 11C-deprenyl-D2 (2), 11C-SL25.1188 (14), 11C-MD230254 (15), dl-4-18F-fluorodeprenyl (16), 6-18F-fluoro-N-methyl-N-(prop-2-yn-1-yl)-hexan-1-amine (17), 18F-Ro 43-0463 (18), and 18F-(S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)-oxazolidin-2-one (19). Among those, only 11C-labeled compounds such as 11C-deprenyl (12), 11C-l-deprenyl-D2, (20) and 11C-SL25.1188 (21) were demonstrated as useful tracers for assessing MAO-B in the human brain. The short half-life of 11C (20.4 min) makes these tracers less suitable for distribution to PET centers not equipped with an on-site cyclotron. So far, no successful 18F-labeled PET radioligand has been validated for clinical use. Therefore, there is keen interest in the development of an 18F-labeled MAO-B inhibitor with a longer half-life and a less reversible kinetic behavior as a molecular imaging biomarker for the detection of MAO-B activity in the brain. Such a radioligand might be a useful imaging tool for the detection of activated astrocytes in central nervous system disorders such as Alzheimer disease, Parkinson disease, and epilepsy. In our recent publications, we have reported several 18F-labeled radiotracers such as 18F-fluorodeprenyl-(N-[(2S)-1-18F-fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine) (22,23), 18F-fluororasagiline (24), (S)-N-(1-18F-fluoro-3-(furan-2-yl)propan-2-yl)-N-methylprop-2-yn-1-amine (25), (S)-1-18F-fluoro-N,4-dimethyl-N-(prop-2-ynyl)pentan-2-amine (25), and 18F-fluororasagiline-D2 (26).

The present work is the continuation of our previous study in which we reported the in vivo evaluation of MAO-B in the cynomolgus monkey brain using 18F-fluorodeprenyl and also investigated its metabolism (23). Quantification of MAO-B using 18F-fluorodeprenyl may not be optimal because its fast, irreversible binding to the enzyme renders the distribution of this radioligand in tissue limited by blood flow rather than by the MAO-B enzyme concentration in regions with high MAO-B activity, as was shown for 11C-deprenyl (20). Indeed, 18F-fluorodeprenyl showed in vivo kinetic behavior similar to that of 11C-deprenyl (20). It has been reported that MAO-B–catalyzed cleavage of the carbon–hydrogen bond of the propargyl group at the carbon is the rate-limiting step in the retention of radioligand in the brain (27). The energy required to cleave the C–D bond is higher than that required to cleave the C–H bond, and the substitution of hydrogen atoms with deuterium atoms reduces the rate of cleavage. The aim of the deuteration is to decrease the affinity to MAO-B through a reduced cleavage rate of the propargyl moiety and thereby improve its sensitivity.

Therefore, our aims in this project were, first, to prepare the precursor and reference standard and develop an efficient synthetic method for labeling a novel bis-deuterium substituted l-deprenyl analog (fluorodeprenyl-D2; Fig. 1) with 18F; second, to characterize its in vitro MAO-B and MAO-A inhibition based on the rate of kynuramine oxidation; third, to evaluate the in vitro autoradiography in postmortem human brain; and fourth, to study the in vivo characteristics by PET measurements in nonhuman primates (NHPs).

FIGURE 1.
FIGURE 1.

Structures of 11C-deprenyl, 18F-fluorodeprenyl, and 18F-fluorodeprenyl-D2 (7).

MATERIALS AND METHODS

Chemistry

The supplemental section details the synthesis of (1,1-2H2)prop-2-yn-1-ol (2), (1,1-2H2)3-bromoprop-1-yne (3), and (S)-2-(((1,1-2H2)-2-ynyl)(methyl)amino)-3-phenyl propan-1-ol (4) (supplemental materials are available at http://jnm.snmjournals.org).

Synthesis of N-(2-Chloro-3-Phenylpropyl)-N-Methyl-[(1,1-2H2)Prop-2-yn-1-Amine] (5a) and (S)-N-(1-Chloro-3-Phenylpropan-2-yl)-N-Methyl-[(1,1-2H2)Prop-2-yn-1-Amine] (5b)

A mixture of 4 (100.0 mg, 0.49 mmol) and triethyl amine (139 μL, 1.0 mmol) in tetrahydrofuran (2 mL) was stirred at room temperature for 30 min. To the stirred mixture, mesylchloride (68.7 mg, 46.4 μL, 0.60 mmol) was added dropwise at −7°C, and the reaction mixture was stirred at room temperature for an additional 30 min. Saturated aqueous Na2CO3 solution (1 mL) was added and stirred for a further 30 min. The organic layer was partitioned between CH2Cl2 (15 mL) and water (15 mL). The organic phase was separated and washed with saturated NaHCO3 solution (15 mL) and brine (15 mL), dried over MgSO4, and filtered. The solvent was removed to obtain the crude product as a light yellow oil. The crude product was purified by silica-gel column chromatography (3:1 hexane:ethyl acetate), and the final product (70 mg, 64%, 0.32 mmol) was obtained as a light yellow oil. The product was analyzed by nuclear magnetic resonance, high-performance liquid chromatography (HPLC), and liquid chromatography–mass spectrometry. The final product was a mixture of 5a (major) and 5b (minor).

Synthesis of (S)-N-(1-Fluoro-3-Phenylpropan-2-yl)-N-Methyl-[(1,1-2H2)Prop-2-yn-1-Amine] (6)

To the stirred solution of 4 (100 mg, 0,49 mmol) in dichloromethane (5 mL), diethylamino sulfur trifluoride (132 μL, 1.0 mmol) was added dropwise at −5°C, and the reaction mixture was stirred for an additional 20 min at the same temperature. Saturated sodium carbonate (2.0 mL) was added to quench unreacted diethylamino sulfur trifluoride. The organic layer was partitioned between CH2Cl2 (25 mL) and water (15 mL). The organic phase was separated and washed with brine (10 mL) and dried over MgSO4 and filtered. The solvent was removed to obtain the crude product as a light yellow oil. The crude product was purified by silica-gel column chromatography (3:1 hexane:ether) and gave the final product (35 mg, 0.17 mmol, 35%). The product was analyzed by nuclear magnetic resonance, HPLC, and liquid chromatography–mass spectrometry.

Radiosynthesis of (S)-N-(1-18F-Fluoro-3-Phenylpropan-2-yl)-N-Methyl-[(1,1-2H2)Prop-2-yn-1-Amine] (18F-Fluorodeprenyl-D2) (7)

18F-fluoride (18F-F−) was produced from a PETtrace cyclotron (GE Healthcare). A dry complex of 18F-F−/K2CO3/K2.2.2 was synthesized following a previously described method (22). The precursor (∼0.02 mmol, ∼5 mg) in dimethyl sulfoxide (500 μL) was added to the dry complex of 18F-F−/K2CO3/K2.2.2. The closed reaction vessel was heated at 140°C for 10 min. The reaction mixture was cooled to room temperature and diluted with water to a total volume of 2 mL before being injected onto the HPLC column for purification. A radioactive fraction corresponding to the pure 7 (retention time, 13–14 min) was collected and diluted with water (50 mL). The resulting mixture was loaded onto a preconditioned SepPak tC18 Plus cartridge (Waters). The cartridge was washed with water (10 mL), and the isolated product was eluted with 1 mL of ethanol into a sterile vial containing phosphate-buffered saline solution (9 mL).

Determination of MAO Inhibition and In Vitro Autoradiography

Human recombinant MAO-B and MAO-A enzymes prepared from insect cells were purchased from Sigma. The assays were designed to determine the inhibition of kynuramine oxidation in the presence of the compounds of interest according to Weissbach et al. (28). The experimental procedure is detailed in the supplemental section.

Nondiseased human brains were obtained from the Department of Forensic and Insurance Medicine, Semmelweis University, Budapest. The brains had been removed during forensic autopsy (control brains) and were handled in a previously described manner (29). Ethical permission was obtained from the relevant Research Ethics Committee of the respective institutions. The experimental procedure is detailed in the supplemental section.

Radiometabolite Analysis and Plasma Protein Binding

A reversed-phase HPLC method was used to determine the percentages of radioactivity corresponding to unchanged radioligand and radiometabolites during the course of a PET measurement. The procedure is detailed in the supplemental section.

Study Design Regarding NHPs, PET Experimental Procedure, and Quantification

Three NHPs—male cynomolgus monkeys (Macaca fascicularis)—were included in the study. The monkeys are owned by the Centre for Psychiatry Research, Department of Clinical Neuroscience, Karolinska Institutet. The study was approved by the Animal Research Ethical Committee of the Northern Stockholm Region Swedish Animal Welfare Agency (diarienummer N386/09 and N452/11). The experiment is detailed in the supplemental section. Altogether, 5 PET measurements of 180 min were performed using a high-resolution research tomograph (Siemens Molecular Imaging) on 3 macaques on separate days. Initially, a baseline measurement was performed for 3 monkeys. On separate days, two of the monkeys underwent a second PET measurement after pretreatment with the MAO-B blocker l-deprenyl 30 min before injection of the radioligand. l-deprenyl was infused intravenously over 10 min, and the selected dose of 1 mg/kg was based on previous PET studies on baboons (30). T1-weighted MR images (128 images; 1.0-mm slices) were acquired on a 1.5-T Signa unit (GE Healthcare) for all monkeys. Volumes of interest were delineated on coregistered MR/PET images, and the delineation was guided by referring to an atlas obtained from 1 rhesus monkey brain. Volumes of interest for caudate, putamen, thalamus; frontal, temporal, parietal, and occipital cortex; medial temporal area; cerebellum; and whole brain were drawn using version 3.3 of PMOD (PMOD Technologies). The MR image was automatically coregistered and resliced to an average PET image using the fusion tool in PMOD. Decay-corrected time–activity curves for all regions plotted over time and radioactivity were expressed as percentage SUV.

All dynamic PET data were quantified with kinetic modeling and compartmental model analysis using the following 3 methods: 1-tissue-compartment model with 2 rate constants (K1, k2) (1-TCM), 2-tissue-compartment model with 4 rate constants (K1, k2, k3, and k4) (2-TCM 4K), and 2-tissue-compartment model with 3 rate constants (K1, k2, and k3) (2-TCM 3K). The following measures were used to directly and indirectly assess the goodness of fit: SE of residuals, the Akaike information criterion, and the Schwartz criterion. The F test was used to assess whether the 2-TCM 4K provided a significantly better fit than the 2-TCM 3K. On the basis of this evaluation and the evident reversibility of the tracer, we selected the 2-TCM 4K as the model of choice and total distribution volume as the outcome measure. Accordingly, we used the revisited Lassen plot (31) to calculate the inhibition that occurred after administration of a well-known MAO-B blocker.

RESULTS

Chemistry and Radiochemistry

The precursor compounds (5a and 5b) and the reference standard (6) were synthesized by standard organic synthesis from the commercially available starting material, propiolic acid (1). The radiolabeling was achieved by nucleophilic substitution of the chloroprecursor mixture of 5a and 5b by 18F-fluoride in the presence of K2.2.2 and K2CO3 as shown in Figure 2. The overall radiosynthesis, including the fluorination reaction, HPLC purification, solid-phase extraction purification, and radiotracer formulation, was completed within 75–80 min. The incorporation yield of the fluorination reactions varied from 45% to 55%, and the radiochemical purity was higher than 99%. The identity of the labeled compound was confirmed by coinjection of the corresponding 19F analog 6 using analytic HPLC. The radioligand 18F-fluorodeprenyl-D2 was found to be stable in phosphate-buffered saline solution (pH 7.4) for the duration of 120 min. 18F-fluorodeprenyl-D2 was obtained with a specific radioactivity of 677 ± 182 GBq/μmol.

FIGURE 2.
FIGURE 2.

Synthesis of precursor (5a + 5b), synthesis of reference standard (6), and radiolabeling of 18F-fluorodeprenyl-D2, (7). Conditions: a, LAD; b, PBr3; c, K2CO3/(1,1-2H2)-1-bromoprop-2-yne; d, pyridine/mesyl chloride; e, diethylaminosulfur trifluoride; f, K18F, K-2,2,2/dimethyl sulfoxide/K2CO3.

In Vitro Inhibition and Autoradiography

Determination of MAO-B and MAO-A inhibition was based on the rate of kynuramine oxidation for the 19F analog of fluorodeprenyl-D2 (6) to determine its specificity for binding to MAO-B. The 50% inhibitory concentration of compound 6 for MAO-B was 227 ± 36.8 nM, and that for MAO-A was more than 50 μM. l-deprenyl and 18F-fluorodeprenyl were used as reference compounds, inhibiting MAO-B with a 50% inhibitory concentration of 13 ± 0.4 and 31 ± 2.2 nM, respectively. Thus, deuteration in compound 6 decreased the 50% inhibitory concentration toward MAO-B by about 7 times as compared with the nondeuterated fluorodeprenyl in the assay used.

The in vitro binding of 18F-fluorodeprenyl-D2 was determined at the coronal and horizontal levels of whole-hemisphere human brain sections obtained from deceased subjects with no sign of any brain disorders. The 18F-fluorodeprenyl-D2 radioligand showed higher selectivity for the MAO-B enzyme than for the MAO-A enzyme, as in vitro binding was almost 100% blocked with the cold ligand l-deprenyl (10 μM), a selective inhibitor of the MAO-B enzyme (Fig. 3). In contrast, 18F-fluorodeprenyl-D2 binding was blocked 12%–20% using the cold compound pirlindole (10 μM), a selective inhibitor of the MAO-A enzyme (Fig. 3). In addition, in vitro baseline binding of 18F-fluorodeprenyl-D2 in whole-hemisphere human brain sections was high in the caudatus, putamen, globus pallidum, and thalamus (brain regions with high MAO-B activity), and lower radioactivity was measured in the cortex and cerebellum.

FIGURE 3.
FIGURE 3.

Autoradiograms of coronal slices in whole-hemisphere human brain with 18F-fluorodeprenyl-D2 (7) at baseline condition and during incubation with pirlindole (10 μM) and l-deprenyl (10 μM).

PET Measurements in Cynomolgus Monkey

The time–activity curves of 18F-fluorodeprenyl-D2 uptake in a cynomolgus monkey brain are shown in Figure 4. 18F-fluorodeprenyl-D2 crossed the blood–brain barrier and bound rapidly, with an average time to peak of 4 min. At baselines, the highest radioactivity concentrations (expressed as SUV) were in the striatum (5.1 ± 1.6) and thalamus (4.6 ± 1.3), followed by the medial temporal area (4.6 ± 2.5). Overall, 18F-fluorodeprenyl-D2 showed fast and constant washout from the brain in subcortical and cortical regions on baseline scans (Fig. 4). The 1-TCM did not provide a good fitting by visual inspection, suggesting the presence of a kinetically distinguished compartment, and was not pursued for further analysis. The 2-TCM 4K estimated the parameters in all baseline scans significantly better than the 2-TCM 3K except for the thalamus of a single NHP. Administration of l-deprenyl (1.0 mg/kg) decreased 18F-fluorodeprenyl-D2 binding in all brain regions and determined a clear change in the slope of the time–activity curves (Fig. 4). The 2-TCM 4K provided, in the pretreatment condition, a statistically significant improvement in fitting of the data in a single NHP. Overall, 4 of 5 experiments did show a better estimation of the parameters with the 2-TCM 4K, providing total distribution volume estimates that could be used as an outcome measure (Table 1). Thereby, MAO-B occupancy of l-deprenyl calculated with the revised Lassen plot was 69% (NHP 2) and 77% (NHP 3). Nondisplaceable volumes of distribution estimated at the x-axis intercept in the Lassen plots were 3.7 and 9.8 mL/cm3, respectively (Fig. 5).

FIGURE 4.
FIGURE 4.

Representative fused PET/MR images of 18F-fluorodeprenyl-D2 (7) and relative SUV time–activity curves under two experimental conditions (baseline and pretreatment) at level of thalamus in 1 NHP.

View this table:
TABLE 1

Total Distribution Values (mL/cm3) Calculated with 2-TCM 4K Under Both Experimental Conditions

FIGURE 5.
FIGURE 5.

Baseline data are used along with occupancy plot to derive occupancy estimates. Data are taken from NHP 2 and NHP 3, which performed both baseline and pretreatment study with l-deprenyl, 1 mg/kg. In both graphs, each data point represents regions of interest included in regression analysis. VND = nondisplaceable volume of distribution.

Radiometabolite Analysis

The radioactivity in arterial blood samples, plasma, and the remaining protein pellet after deproteinization of plasma with acetonitrile was measured by a well counter. Recovery of radioactivity from plasma into acetonitrile was more than 85%. The plasma obtained from arterial blood samples taken at various time points after the injection of 18F-fluorodeprenyl-D2 was analyzed by radio-HPLC. All detected radiometabolites were less lipophilic than the radioligand. The most lipophilic radiometabolite, eluting with a retention time of 7.9 min closely before the radioligand retention time, 8.6 min, was identified as 18F-fluorometamphatamine-D2 (Fig. 6A) by comparing its retention time with that of the synthesized reference compound. Approximately 5% of the total radioactivity originated from 18F-fluorometamphetamine-D2 during the PET scan, as shown in Figure 6B.

FIGURE 6.
FIGURE 6.

(A) Radiochromatogram of plasma taken 30 min after injection of radioligand. (B) In vivo metabolism of 18F-fluorodeprenyl-D2 (7) shown as relative plasma composition of parent compound and identified radiometabolite 18F-flurometamphatamine-D2 in total plasma radioactivity during 180 min of PET scan. Values are average of measurements performed on 3 cynomolgus monkeys. a.u. = arbitrary unit.

Protein binding of 18F-fluorodeprenyl-D2 was measured using ultrafiltration, and an average of 33.8% ± 4.1% of the radioligand was in protein-free form.

DISCUSSION

It has been shown that the dideuterated derivative of the MAO-B inhibitor deprenyl can be used as a marker for the density of astrocytes in the brain (32,33). In the present study we show the potential of 18F-fluorodeprenyl-D2 to visualize MAO-B activity in vivo.

Compound 2 was synthesized by treating propiolic acid with lithium aluminum deuteride. The key parameter of this reaction was to keep the temperature below −50°C. In the second step, 2 was brominated with PBr3 to synthesize 3. Compound 4 was synthesized from 3 and (4R,5R)-4-methyl-5-phenyl-3-(prop-2-ynyl)oxazolidin-2-one following a previously described procedure (22). A mixture of two chlorinated isomers, 5a and 5b, was formed from 4 on treatment with mesylchloride. Fluoride 6 was also synthesized from amino alcohol 4 by fluorination with diethylamino sulfur trifluoride. The formation of regioisomeric chlorides 5a and 5b can be explained by an intermediate aziridinium ion resulting from an intramolecular nucleophilic substitution attack of the free electron pair of the nitrogen, as has been previously described elsewhere (34). Radiolabeling was done by a 1-step nucleophilic substitution reaction in which chloride was substituted by 18F-fluoride. The synthesis resulted in two isomers of 18F-labeled products, which were purified by a semipreparative HPLC column.

High in vitro 18F-fluorodeprenyl-D2 binding was measured in several brain regions with high MAO-B activity, and l-deprenyl (10 μM) successfully displaced all 18F-fluorodeprenyl-D2 binding. In NHPs in vivo, 18F-fluorodeprenyl-D2 showed favorable kinetic properties, with relatively steady washout from the brain in most experiments and conditions. The deuteration seemed, in vivo, to lead to a further reduction in covalent binding of the tracer to the enzyme, resulting in a less irreversible behavior even when it was compared, in the same experimental condition and NHP, with the 11C-deprenyl-D2 kinetic (Fig. 7 and supplemental section). In the present study, a more reversible kinetic of 18F-fluorodeprenyl-D2 was confirmed by model comparison. The 2-TCM suitable for irreversible tracers (2-TCM 3K) did not perform best in terms of fitting in most experiments; hence, the 2-TCM 4K was the most appropriate model to describe the brain kinetic of 18F-fluorodeprenyl-D2. Consequently, estimation of total distribution volume was considered a more reliable outcome measure to evaluate the availability of MAO-B in NHPs. Moreover, estimates of nondisplaceable volume of distribution obtained from the revised Lassen plot allowed for measurement of the level of specific binding, which in the case of a high-density MAO-B region such as the thalamus (total distribution volumes of 34.91 mL/cm3 on average) was quite high (79% on average). The calculated occupancy after pretreatment with deprenyl (1 mg/kg) was higher (77% vs. 61.7%) than a previous estimation obtained with 11C-deprenyl-D2 (Supplemental Table 3).

FIGURE 7.
FIGURE 7.

Head-to-head comparison of two baseline scans performed under same experimental condition on the same NHP (NHP 3) with 11C-deprenyl-D2 (A) and 18F-fluorodeprenyl-D2 (B). cx = cortex.

It has been reported that the two main radiometabolites of 11C-deprenyl are 11C-CO2 and 11C-metamphetamine (35) and that at 30 min after injection, 30% of the total radiometabolites found in baboon plasma is in the form of 11C-CO2. In the case of 18F-fluorodeprenyl-D2, there is no possibility of having radiolabeled CO2; 18F-fluorometamphetamine-D2 is more likely. The potential specific contribution of one of the most lipophilic radiometabolites is questionable. Indeed, one of the identified radiometabolites, 18F-fluorometamphetamine-D2, was observed at less than 5% on average and in some NHPs was not separated on the HPLC because of its low amount. A structurally similar radiotracer has been shown to enter the human brain and bind primarily to monoamine transporters (36).

The presence of such an amount of 18F-fluorometamphetamine-D2 in the plasma might contribute to a small increase in nonspecific binding of 18F-fluorodeprenyl-D2 in the brain. Future challenges would include optimization of the HPLC conditions to achieve a better separation of radiometabolites in plasma and to investigate blood–brain barrier–penetrating radiometabolites using mouse models.

CONCLUSION

In the present work, an efficient synthesis strategy for an 18F-labeled deuterium substituted fluoro-analog of l-deprenyl was established, yielding the target compound. In vitro, MAO inhibition demonstrated a moderate binding affinity to recombinant MAO-B. In vitro autoradiography demonstrated selective binding to brain regions, such as the striatum, containing MAO-B. In vivo characteristics in a cynomolgus monkey showed moderate uptake in brain regions known to be rich in MAO-B. The deuterated analog of 18F-fluorodeprenyl is more stable in monkey blood plasma than the nondeuterated analog. In addition, the deuteration reduced the rate of radioligand trapping in the monkey brain, leading to improved sensitivity. The PET studies were confirmed by the kinetic analysis performed on NHPs. A faster kinetic and the fluorine labeling are possible advantages over other, previously developed, MAO-B imaging radioligands such as 11C-D2-deprenyl. Together, these results suggest that 18F-fluorodeprenyl-D2 is an improved PET radioligand for visualization of MAO-B activity in the human brain.

DISCLOSURE

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734. This research received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement HEALTHF2-2011-278850 (INMiND). No other potential conflict of interest relevant to this article was reported.

Acknowledgments

We thank all the members of Karolinska Institutet PET Centre.

Footnotes

  • Published online Nov. 19, 2015.

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  • Received for publication May 19, 2015.
  • Accepted for publication October 19, 2015.
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