Synthesis and characterization of EADAM: a selective radioligand for mapping the brain serotonin transporters by positron emission tomography

[11C]N,N-Dimethyl-2-(2'-amino-4'-ethylphenylthio)benzylamine ([11C]EADAM) was synthesized in the development of a serotonin transporter (SERT) imaging ligand for positron emission tomography (PET). The methods of ligand synthesis, results of in vitro characterization, 11C labeling and in vivo micro-PET imaging studies of [11C]EADAM in cynomolgus monkey brain are described. 11C was introduced into N,N-dimethyl-2-(2'-amino-4'-ethylphenylthio)benzylamine (5) by alkylation of N-methyl-2-(2'-amino-4'-ethylphenylthio)benzylamine (10) in 32% radiochemical yield (end of bombardment [EOB], decay-corrected from [11C]methyl iodide). Competition binding assays in cells stably expressing the transfected human dopamine transporter (DAT), SERT and norepinephrine transporter (NET) labeled with [3H]WIN 35428 or [(125)I]RTI-55, [3H]citalopram and [3H]nisoxetine, respectively, indicated the following order of SERT affinity: ADAM>EADAM>fluvoxamine. The affinity of EADAM for DAT and NET was 500- and >1000-fold lower, respectively, than for SERT. Micro-PET brain imaging studies in a cynomolgus monkey demonstrated high [11C]EADAM uptake in the striatum, thalamus and brainstem. [11C]EADAM uptake in these brain regions peaked in less than 60 min following administration of [11C]EADAM. The tissue-to-cerebellum ratios of the striatum, thalamus and brainstem were 1.67, 1.71 and 1.63, respectively, at 120 min postinjection of [11C]EADAM. Analysis of monkey arterial plasma samples using high-pressure liquid chromatography determined there was no detectable formation of lipophilic radiolabeled metabolites capable of entering the brain. In a displacement experiment with citalopram in a cynomolgus monkey, radioactivity in the striatum, thalamus and brainstem was displaced 20-60 min after administration of citalopram. In a blocking experiment with citalopram in a cynomolgus monkey, radioactivity in the striatum, thalamus and brainstem was significantly reduced. These results support the candidacy of [11C]EADAM as a radioligand for visualizing brain SERT using PET.     

PET examination of [(11)C]5-methyl-6-nitroquipazine,a radioligand for visualization of the serotonin transporter

Radiohalogenated 5-halo-6-nitroquipazine analogues have been shown to be potential radioligands for visualization of the serotonin transporter (5-HTT) with PET and SPECT. In the present study a methylated analogue, 5-methyl-6-nitroquipazine (MNQP), was radiolabeled with carbon-11 in a two step reaction via a palladium catalyzed cross coupling reaction between N-t-BOC-protected 5-tributylstannyl-6-nitroquipazine and [(11)C]methyl iodide as key step. [(11)C]MNQP was examined in the cynomolgus monkey brain with positron emission tomography (PET) and the appearance of labeled metabolites in monkey plasma was measured with gradient HPLC. Radioactivity increased continuously in all brain regions during the 90 minutes acquisition time. Highest accumulation of radioactivity was observed in the thalamus and brainstem, regions with a known high density of 5-HTT. The calculated ratios between the thalamus and brainstem to the 5-HTT poor cerebellum were 1.5 and 1.3-1.4, respectively, 80 minutes after radioligand injection. Pretreatment with citalopram prior to the PET measurement markedly reduced the binding in the thalamus and the brainstem. At 15 and 30 minutes after injection of [(11)C]MNQP approximately 90% and 60%, respectively, of radioactivity in plasma represented unchanged radioligand. The slow kinetics and moderate ratios recorded however, may limit its use as a PET radioligand for quantitative studies of the serotonin transporter with PET.     

Synthesis and pharmacological characterization of a new PET ligand for the serotonin transporter: [11C]5-bromo-2-[2-(dimethylaminomethylphenylsulfanyl)]phenylamine ([11C]DAPA)

A new PET radioligand for the serotonin transporter (SERT), [11C]-5-bromo-2-[2-(dimethylaminomethylphenylsulfanyl)]phenylamine ([11C]DAPA (10), was synthesized and evaluated in vivo in rats and baboons. [11C]DAPA (10) was prepared from its monomethylamino precursor 8 by reaction with high specific activity [11C]methyl iodide. Radiochemical yield was 24 +/- 5% based on [11C]methyl iodide at end of bombardment (EOB, n = 10) and specific activity was 1,553 +/- 939 Ci/mmol at end of synthesis (EOS, n = 10). Binding assays indicated that [11C]DAPA displays high affinity (Ki 1.49 +/- 0.28 nM for hSERT) and good selectivity for the SERT in vitro. Biodistribution studies in rats indicated that [11C]DAPA enters into the brain readily and localizes in brain regions known to contain high concentrations of SERT, such as the thalamus, hypothalamus, frontal cortex and striatum. Moreover, such binding in SERT-rich regions of the brain are blocked by pretreatment with either the selective serotonin reuptake inhibitor (SSRI) citalopram and by the cold compound itself, demonstrating that [11C]DAPA binding in the rat brain is saturable and specific to SERT. Imaging experiments in baboons indicated that [11C]DAPA binding is consistent with the known distribution of SERT in the baboon brain, with highest levels of radioactivity detected in the midbrain and thalamus, intermediate levels in the hippocampus and striatum, and lower levels in the cortical regions. Pretreatment of the baboon with citalopram 10 min before radioactivity injection blocked the binding of [11C]DAPA in all brain regions that contain SERT. Kinetic analysis revealed that, in all brain regions examined, [11C]DAPA specific to nonspecific distribution volume ratios (V(3)") are higher than [11C](+)-McN 5652 and similar to [11C]DASB. In summary, [11C]DAPA appears to be a promising radioligand suitable for the visualization of SERT in vivo using PET.     

Synthesis and evaluation of racemic [(11)C]NS2456 and enantiomers as selective serotonin reuptake radiotracers for PET

Positron emission tomography (PET) radiotracers are needed for quantifying serotonin uptake sites in the living brain. Therefore, we evaluated a new selective serotonin reuptake inhibitor, NS2456, to determine whether it is suited for use in PET. Racemic NS2456 [(1RS,5SR)-8-methyl-3-[4-trifluoromethoxyphenyl]-8-azabicyclo [3.2.1]oct-2-ene] and its N-demethylated analog, racemic NS2463, selectively inhibited serotonin uptake in rat brain synaptosomes; their IC(50) values were 3000-fold lower for [(3)H]serotonin than for either [(3)H]dopamine or [(3)H]noradrenaline. The enantiomers of NS2463 were also potent inhibitors of serotonin uptake in vitro, but they failed to show stereoselectivity. Racemic NS2463 as well as its enantiomers were radiolabelled by N-methylation with C-11, yielding [(11)C]NS2456 for use in PET of the living porcine brain. The compounds crossed the blood-brain barrier rapidly and accumulated preferentially in regions rich in serotonin uptake sites (e.g., brainstem, subthalamus and thalamus). However, their binding potentials were relatively low and no stereoselectivity was found. Thus, neither racemic [(11)C]NS2456 nor its [(11)C]-labelled enantiomers are ideal for PET neuroimaging of neuronal serotonin uptake sites.     

A PET imaging agent with fast kinetics: synthesis and in vivo evaluation of the serotonin transporter ligand [11C]2-[2-dimethylaminomethylphenylthio)]-5-fluorophenylamine ([11C]AFA)

A new serotonin transporter (SERT) ligand, [11C]2-[2-(dimethylaminomethylphenylthio)]-5-fluorophenylamine (10, [11C]AFA), was synthesized and evaluated as a candidate PET radioligand in pharmacological and pharmacokinetic studies. As a PET radioligand, AFA (8) can be labeled with either C-11 or F-18. In vitro, AFA displayed high affinity for SERT (Ki 1.46 +/- 0.15 nM) and lower affinity for norepinephrine transporter (NET, Ki 141.7 +/- 47.4 nM) or dopamine transporter (DAT, Ki > 10,000 nM). [11C]AFA (10) was prepared from its monomethylamino precursor 9 by reaction with high specific activity [11C]methyl iodide. Radiochemical yield was 43 +/- 20% based on [11C]methyl iodide at end of bombardment (EOB, n = 10) and specific activity was 2,129 +/- 1,369 Ci/mmol at end of synthesis (EOS, n = 10). Biodistribution studies in rats indicated that [11C]AFA accumulated in brain regions known to contain high concentrations of SERT. Binding in SERT-rich brain regions was reduced significantly by pretreatment with either the cold compound 8 or with the selective serotonin reuptake inhibitor (SSRI) citalopram, but not by the selective norepinephrine reuptake inhibitor nisoxetine, thus underlining its in vivo binding selectivity and specificity for SERT. Imaging experiments in baboons demonstrated that the uptake pattern of [11C]AFA in the baboon brain is consistent with the known distribution of SERT, with highest activity levels in the midbrain and thalamus, followed by striatum, hippocampus, and cortical regions. Activity levels in the baboon brain peaked at 15-40 min after radioligand injection, indicating a fast uptake kinetics for [11C]AFA. Pretreatment of the baboon with citalopram (4 mg/kg) significantly reduced the specific binding of [11C]AFA in all SERT-containing brain regions. Kinetic analysis revealed that the regional equilibrium specific to non-specific partition coefficients (V3") of [11C]AFA are similar to those of [11C]McN5652, but lower than those of [11C]AFM or [11C]DASB. In summary, [11C]AFA appears to be an appropriate PET radioligand with a fast brain uptake kinetics:     

Kinetics of [11C]N,N-dimethylphenylethylamine in mice and humans: potential for measurement of brain MAO-B activity

Carbon-11-labeled N,N-dimethylphenylethylamine ([11C]DMPEA) was synthesized by the reaction of N-methylphenylethylamine with [11C]methyl iodide. This newly synthesized radiotracer was developed for the purpose of in vivo measurement of monoamine oxidase-B activity in the brain using a metabolic trapping method. Initially, biodistribution was investigated in mice. The rapid and high uptake of 11C radioactivity in the brain was observed following intravenous injection of [11C]DMPEA, the peak of which was reached at 1 min, followed by a decrease at 1-5 min and slowly thereafter. The kinetics of [11C]DMPEA in the human brain were determined using positron emission tomography (PET) and showed that 11C radioactivity increased gradually over 60 min following initial rapid uptake of 11C radioactivity, with basal ganglia and thalamus showing high accumulation.     

Characterization of bromine-76-labelled 5-bromo-6-nitroquipazine for PET studies of the serotonin transporter.

The development of suitable radioligands for brain imaging of the serotonin transporter is of great importance for the study of depression and other affective disorders. The potent and selective serotonin transporter ligand, 5-iodo-6-nitro-2-piperazinylquinoline, has been labelled with iodine-123 and used as a radioligand for single photon emission computerized tomography. To evaluate the potential of the bromine-76-labelled analogue, 5-bromo-6-nitroquipazine, as a radioligand for positron emission tomography (PET), its brain distribution and binding characteristics were examined in rats. In vivo brain distribution and ex vivo autoradiography demonstrated that [76Br]5-bromo-6-nitroquipazine enters the brain rapidly. The regional brain distribution of [76Br]5-bromo-6-nitroquipazine was consistent with the known distribution of serotonin transporters in the midbrain, pons, thalamus, striatum, and neocortex. Specific binding was inhibited by the selective serotonin reuptake inhibitor citalopram. The peripheral metabolism in plasma was rapid, but more than 90% of the radioactivity in brain represented unchanged radioligand 2 h postinjection (p.i.). A preliminary PET study was also performed in a baboon. Following the intravenous injection of [76Br]5-bromo-6-nitroquipazine in a baboon, there was a conspicuous accumulation of radioactivity in thalamus, striatum, and pons. The radioactivity in these brain regions was 1.5 times higher than in the cerebellum at 3 h and 2.5-4 times higher at 24 h. A rapid metabolism of the radioligand in plasma was observed (38% unchanged after 5 min). The results indicate that [76Br]5-bromo-6-nitroquipazine has potential for PET imaging of the serotonin transporter.     

11C]SMe-ADAM, an imaging agent for the brain serotonin transporter: synthesis, pharmacological characterization and microPET studies in rats

N,N-Dimethyl-2-(2-amino-4-methylthiophenylthio)benzylamine (SMe-ADAM, 1) is a highly potent and selective inhibitor of the serotonin transporter (SERT). This compound was labeled with carbon-11 by methylation of the S-desmethyl precursor 10 with [(11)C]methyl iodide to obtain the potential positron emission tomography (PET) radioligand [(11)C]SMe-ADAM. The radiochemical yield was 27 +/- 5%, and the specific radioactivity was 26-40 GBq/micromol at the end of synthesis. Ex vivo and in vivo biodistribution experiments in rats demonstrated a rapid accumulation of the radiotracer in brain regions known to be rich in SERT, such as the thalamus/hypothalamus region (3.59 +/- 0.41%ID/g at 5 min after injection). The specific uptake reached a thalamus to cerebellum ratio of 6.74 +/- 0.95 at 60 min postinjection. The [(11)C]SMe-ADAM uptake in the thalamus was significantly decreased by pretreatment with fluoxetine to 38 +/- 11% of the control value. Furthermore, no metabolites of [(11)C]SMe-ADAM could be detected in the SERT-rich regions of the rat brain. It is concluded that [(11)C]SMe-ADAM may be a suitable PET ligand for SERT imaging in the living brain.     

PET imaging of dopamine transporters in the human brain using [(11)C]-beta-CPPIT, a cocaine derivative lacking the 2 beta-ester function.

The compound 3 beta-(4'-chlorophenyl)-2 beta-(3'-phenylisoxazol-5'-yl)tropane (CPPIT or RTI 177) is a 2beta-heterocyclic substituted cocaine congener with high in vitro selectivity and affinity for the dopamine transporter relative to serotonin and norepinephrine transporters. The aim of the present study was to evaluate the in vivo selectivity of [(11)C]-beta-CPPIT and to determine whether [(11)C]-beta-CPPIT may be a suitable alternative to existing DAT PET radioligands. [(11)C]-beta-CPPIT was prepared by N-alkylation of the free amine with [(11)C]methyl iodide. In mouse brain, the striatal binding of [(11)C]-beta-CPPIT was reduced significantly by preinjecting the dopamine reuptake antagonist GBR 12909 (5 mg/kg). By contrast, radioactivity uptake in the brain was not affected significantly by the preinjection of citalopram (5 mg/kg) and desipramine (5 mg/kg), inhibitors for the serotonin and norepinephrine transporters, respectively. No effect was also observed by pretreatment with ketanserin (2.5 mg/kg) a compound with high affinity for the 5-HT(2A)-receptor and the vesicular monoamine transporter. In a PET study with six healthy volunteers high striatal uptake was observed. The distribution pattern of [(11)C]-beta-CPPIT was similar to the known distribution of the dopamine transporter in the human brain. Compared to (123)I labeled beta-CIT, the rate of metabolic degradation of [(11)C]-beta-CPPIT was almost twofold slower suggesting that bioisosteric heterocyclic substitution of the ester group at the 2 beta-position of the tropane ring does have an influence on the rate of metabolism of [(11)C]-beta-CPPIT. The rank order of the distribution volumes obtained via the one-tissue compartment model is also similar to the reported distribution of DAT. These preliminary results suggest that [(11)C]-beta-CPPIT may be a useful PET radioligand for the visualization and quantification of dopamine transporters in man.     

PET evaluation of a tetracyclic, atypical antidepressant, [N-methyl-11C]mianserin, in the living porcine brain

We synthesized [N-methyl-11C]mianserin by alkylation of N-desmethyl mianserin with [11C]methyl iodide followed by HPLC purification. We used PET for determining the regional cerebral pharmacokinetics of the radiotracer in anesthetized swine. [N-methyl-11C]Mianserin entered most brain regions readily (range of K1 values: 0.66-1.13), reaching highest levels in the basal ganglia and thalamus. The binding potential of [N-methyl-11C]mianserin was relatively low (range: 0.07-0.50), but regional differences were nonetheless observed, with highest values in the temporal cortex and lowest values in the brainstem. These PET findings, which are the first ones for a tetracyclic, antidepressant drug, show that [N-methyl-11C]mianserin has only a limited degree of regional specificity of binding in the living brain.     

Synthesis and in vivo evaluation of novel radiotracers for the in vivo imaging of the norepinephrine transporter

The (R,R) and (S,S) enantiomers of 2-[(2-methoxyphenoxy)phenylmethyl]morpholine (MeNER) have been radiolabelled with carbon-11 in good yield and at high specific activity. These radiotracers are close analogues of reboxetine, a potent and selective ligand for the norepinephrine transporter (NET). They were examined as potential ligands for imaging NET in vivo by positron emission tomography (PET). The in vivo brain distribution of both [(11)C]-labeled enantiomers were evaluated in rats. Following tail-vein injection of the (R,R)-enantiomer regional brain uptake and washout of radioactivity was homogeneous at all time points examined (5-60 min). In contrast, administration of the (S,S)-enantiomer produced a heterogeneous distribution of radioactivity in brain with highest uptake in the hypothalamus, a NET rich region, and lowest uptake in the striatum, a brain region devoid of NET. Hypothalamus to striatum ratios of 2.5 to one were achieved at 60 min post injection of (S,S)-[(11)C]-MeNER. Pre-injection of the norepinephrine reuptake inhibitors, reboxetine or desipramine, reduced hypothalamus to striatum ratios to near unity while reuptake inhibitors of dopamine and serotonin had no significant effect on binding. In vitro autoradiography studies (rat brain slices) with (S,S)-[(11)C]-MeNER produced a regional distribution pattern that was consistent with the reported distribution of NET. (S,S)-[(11)C]-MeNER has the potential to be the first successful PET ligand to image NET.     

New halogenated [11C]WAY analogues, [11C]6FPWAY and [11C]6BPWAY--radiosynthesis and assessment as radioligands for the study of brain 5-HT1A receptors in living monkey

[Carbonyl-(11)C]WAY-100635 ([(11)C]WAY) is an established radioligand for the study of brain serotonin(1A) (5-HT(1A)) receptors in living animals and humans with positron emission tomography (PET). There is a recognised need to develop halogenated ligands for 5-HT(1A) receptors, either for labelling with longer-lived fluorine-18 for more widespread application with PET or with iodine-123 for application with single photon emission tomography (SPET). Here we used autoradiography and PET to assess two new halogenated analogues of WAY, namely 6BPWAY and 6FPWAY [N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl)ethyl))-N-(2-(6-bromo-/fluoro-pyridinyl))cyclohexanecarboxamide] as prospective radioligands, initially using carbon-11 as the radiolabel. Labelling of 6BPWAY and 6FPWAY with carbon-11 was accomplished by acylation of the corresponding secondary amine precursors with [carbonyl-(11)C]cyclohexanecarbonyl chloride. After incubation of human brain crysections with [(11)C]6BPWAY or [(11)C]6FPWAY, the highest accumulation of radioactivity was observed in cortical areas and the hippocampal formation. Both radioligands had high nonspecific binding. There was a rapid accumulation of radioactivity in the monkey brain after intravenous injection of [(11)C]6BPWAY and [(11)C]6FPWAY. High accumulation of radioactivity was observed in the frontal and temporal cortex and the raphe nuclei, areas known to contain a high density of 5-HT(1A) receptors. The ratios of radioactivity in receptor-rich temporal cortex to that in receptor-poor cerebellum at peak equilibrium were 1.9 (at 10 min) and 3.0 at (at 20 min) for [(11)C]6BPWAY and [(11)C]6FPWAY, respectively. In pretreatment experiments with high doses of unlabelled WAY, the level of radioactivity in the frontal and temporal cortex and the raphe nuclei was reduced to the same level as in the cerebellum. Radioactive metabolites of [(11)C]6FPWAY appeared at a rate similar to those for [(11)C]WAY, with 17% of the radioactivity in plasma represented by unchanged radioligand after 40 min. Radioactive metabolites of [(11)C]6BPWAY appeared much more slowly. At 40 min after injection 45% of the radioactivity in plasma still represented unchanged radioligand. The results indicate that 6-pyridinyl radiohalogented analogues of WAY are new leads to radioligands for PET or SPET.     

The new PET imaging agent [11C]AFE is a selective serotonin transporter ligand with fast brain uptake kinetics

A new positron emission tomography (PET) radioligand for the serotonin transporter (SERT), [¹¹C]2-[2-[[(dimethylamino)methyl]phenyl]thio]-5-(2-fluoroethyl)phenylamine ([¹¹C]AFE, 12), was synthesized and evaluated in vivo in rats and baboons. [¹¹C]AFE (12) was prepared from its monomethylamino precursor 11 by reaction with high specific activity [¹¹C]methyl triflate. Radiochemical yield was 32±17% based on [¹¹C]methyl triflate (n=6) and specific activity was 1670±864 Ci/mmol at end of synthesis (EOS, n=6). Binding assays indicated that AFE displays high affinity for SERT (K_i=1.80 nM for hSERT) and lower affinity for norepinephrine transporter (K_i=946 nM for hNET) or dopamine transporter (K_i>10,000 nM for hDAT). In addition, AFE displays negligible binding affinities for other serotonin and dopamine receptors, indicating an excellent binding selectivity in vitro. Biodistribution studies in rats indicated that [¹¹C]AFE enters the brain readily and localizes in regions known to contain high concentrations of SERT, such as the thalamus, hypothalamus, frontal cortex and striatum. Moreover, such binding in SERT-rich brain regions is reduced significantly by pretreatment with either citalopram or the cold compound itself, but not by nisoxetine or GBR 12935, thus demonstrating that [¹¹C]AFE binding in the rat brain is saturable, specific and selective for the SERT. Imaging experiments in baboons indicated that the uptake pattern of [¹¹C]AFE is consistent with the known distribution of SERT in the baboon brain, with high levels of radioactivity detected in the midbrain and thalamus, moderate levels in the hippocampus and striatum and low levels in the cortical regions. The uptake kinetics of [¹¹C]AFE in the baboon brain is rapid, with activity in the midbrain and thalamus peaking at 15–40 min postinjection. Pretreatment of the baboon with citalopram (4 mg/kg) 20 min before radioactivity injection reduced the binding of [¹¹C]AFE in all SERT-containing brain regions to the level in the cerebellum. Kinetic analysis revealed that in all brain regions examined, [¹¹C]AFE specific-to-nonspecific partition coefficients (V₃″) are similar to those of [¹¹C]McN5652 and [¹¹C]2-[2-[[(dimethylamino)methyl]phenyl]thio]-5-fluorophenylamine ([¹¹C]AFA), but lower than those of [¹¹C]2-[2-[[(dimethylamino)methyl]phenyl]thio]-5-fluoromethylphenylamine ([¹¹C]AFM) or [¹¹C]DASB. In summary, [¹¹C]AFE appears to be a PET radioligand with fast brain uptake kinetics and can be used for the visualization and quantification of SERT in vivo.     

Carbon-11 epidepride: a suitable radioligand for PET investigation of striatal and extrastriatal dopamine D2 receptors.

Epidepride [(S)-(-)-N-([1-ethyl-2-pyrrolidinyl]methyl)-5-iodo-2,3-dimethoxybenza mide] binds with a picomolar affinity (Ki = 24 pM) to the dopamine D2 receptor. Iodine-123-labeled epidepride has been used previously to study striatal and extrastriatal dopamine D2 receptors with single photon emission computed tomography (SPECT). Our aim was to label epidepride with carbon-11 for comparative quantitative studies between positron emission tomography (PET) and SPECT. Epidepride was synthesized from its bromo-analogue FLB 457 via the corresponding trimethyl-tin derivative. In an alternative synthetic pathway, the corresponding substituted benzoic acid was reacted with the optically pure aminomethylpyrrolidine-derivative. Demethylation of epidepride gave the desmethyl-derivative, which was reacted with [11C]methyl triflate. Total radiochemical yield was 40-50% within a total synthesis time of 30 min. The specific radioactivity at the end of synthesis was 37-111 GBq/micromol (1,000-3,000 Ci/mmol). Human postmortem whole-hemisphere autoradiography demonstrated dense binding in the caudate putamen, and also in extrastriatal areas such as the thalamus and the neocortex. The binding was inhibited by unlabeled raclopride. PET studies in a cynomolgus monkey demonstrated high uptake in the striatum and in several extrastriatal regions. At 90 min after injection, uptake in the striatum, thalamus and neocortex was about 11, 4, and 2 times higher than in the cerebellum, respectively. Pretreatment experiment with unlabeled raclopride (1 mg/kg) inhibited 50-70% of [11C]epidepride binding. The fraction of unchanged [11C]epidepride in monkey plasma determined by a gradient high performance liquid chromatography (HPLC) method was about 30% of the total radioactivity at 30 min after injection of [11C]epidepride. The availability of [11C]epidepride allows the PET-verification of the data obtained from quantitation studies with SPECT.     

A new positron emission tomography imaging agent for the serotonin transporter: synthesis, pharmacological characterization, and kinetic analysis of [11C]2-[2-(dimethylaminomethyl)phenylthio]-5-fluoromethylphenylamine ([11C]AFM)

The synthesis, radiolabeling, and in vitro and in vivo evaluation of a new positron emission tomography (PET) radioligand for the serotonin transporter (SERT), [(11)C]2-[2-(dimethylaminomethyl)phenylthio]-5-fluoromethylphenylamine ([(11)C]AFM) is reported. AFM was prepared from 4-chloro-3-nitrobenzyl acetate and thiosalicylic acid in a five-step synthetic sequence. In binding studies in vitro with cloned human transporters, AFM displayed high binding affinity (Ki 1.04 nmol/L for hSERT) and good selectivity (Ki 664 nmol/L for hNET and >10,000 nmol/L for hDAT) for SERT. The radiolabled compound [(11)C]AFM was prepared in 30-37 minutes from its monomethylamine precursor by reaction with high specific activity [(11)C]iodomethane. Radiochemical yield was 12.3 +/- 8.1% based on [(11)C]iodomethane and specific activity was 1733 +/- 428 Ci/mmol at end of synthesis (EOS, n = 14). Radiochemical and chemical purity of the final product was >97%. Biodistribution studies in rats indicated that [(11)C]AFM entered the brain readily and localized in regions known to contain high concentrations of SERT, with high specific to nonspecific binding ratios. Furthermore, binding of [(11)C]AFM in SERT-rich regions was blocked by the cold compound AFM and the selective serotonin reuptake inhibitor citalopram but not by the selective norepinephrine reuptake inhibitor nisoxetine or the selective dopamine reuptake inhibitor GBR 12935. At 30 minutes after injection, >95% of the brain activity corresponded to the parent compound, indicating the absence of radiolabeled metabolites in the rat brain. PET imaging experiments in baboons showed a brain distribution pattern of [(11)C]AFM consistent with the regional concentrations of SERT, with the highest levels of radioactivity detected in the midbrain and thalamus, moderate levels in the hippocampus and striatum, and the low levels in the cortical regions. Pretreatment of the baboons with citalopram (4 and 6 mg/kg, intravenously) reduced regional brain distribution volumes to low and homogeneous levels, thus underlining the binding specificity of [(11)C]AFM for SERT in vivo. Analysis of blood samples indicated a fast metabolism of the radioligand into more hydrophilic components, as well as the absence of radiolabeled lipophilic metabolites. Regional time-activity curves were analyzed with kinetic and graphical analysis methods using the arterial concentrations as input function. Both methods returned similar kinetic parameters and documented high specific to nonspecific equilibrium coefficients (V(3)") for [(11)C]AFM. Identical V(3)" values were also derived with the simple reference tissue method, indicating that quantification of SERT with [(11)C]AFM can be achieved without arterial blood sampling. In summary, [(11)C]AFM appears to be an excellent PET radioligand for the visualization and reliable quantification of SERT in vivo.     

Specific in vivo binding to the norepinephrine transporter demonstrated with the PET radioligand, (S,S)-[11C]MeNER

(S,S)-2-(alpha-(2-Methoxyphenoxy)benzyl)morpholine (MeNER), an O-methyl analog of the selective and potent norepinephrine transporter (NET) inhibitor, (S,S)-reboxetine, and its less active enantiomer, (R,R)-MeNER, have each been radiolabeled by O-methylation of their corresponding phenolic precursors in good yields from [(11)C]methyl iodide or [(11)C]methyl triflate. Radiochemical purities were >99% and specific radioactivity at time of injection was about 74 GBq/micromol. Autoradiographic examination of (S,S)-[(11)C]MeNER binding to human brain slices post mortem indicated specific binding in a brain region including the locus coeruleus. PET examination of both [(11)C]MeNER enantiomers in a cynomolgus monkey demonstrated a higher specific binding of the (S,S)-enantiomer with ratios of 1.4-1.6 in the lower brainstem, mesencephalon and thalamus to striatum. Pretreatment with the NET ligand, desipramine, decreased the specific binding of (S,S)-[(11)C]MeNER. Labeled metabolites of [(11)C]MeNER were all more polar. (S,S)-[(11)C]MeNER is a good lead compound in the search for a selective radioligand for quantitation of NET in the human brain in vivo.     

Imaging the serotonin transporter with positron emission tomography: initial human studies with [11C]DAPP and [11C]DASB

Two novel radioligands, N,N-dimethyl-2-(2-amino-4-methoxyphenylthio) b enzylamine (DAPP) and (N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine (D ASB), were radiolabeled with carbon-11 and evaluated as in vivo probes of the serotonin transporter (SERT) using positron emission tomography (PET). Both compounds are highly selective, with nanomolar affinity for the serotonin transporter and micromolar affinity for the dopamine and norepinephrine transporters. Six volunteers were imaged twice, once with each of the two radioligands. Both ligands displayed very good brain penetration and selective retention in regions rich in serotonin reuptake sites. Both had similar brain uptake and kinetics, but the cyano analogue, [11C]DASB, had a slightly higher brain penetration in all subjects. Plasma analysis revealed that both radiotracers were rapidly metabolized to give mainly hydrophilic species as determined by reverse-phase high-performance liquid chromatography. Inhibition of specific binding to the SERT was demonstrated in three additional subjects imaged with [11C]DASB following an oral dose of the selective serotonin reuptake blocker citalopram. These preliminary studies indicate that both these substituted phenylthiobenzylamines have highly suitable characteristics for probing the serotonin reuptake system with PET in humans.     

18F-labeled FECNT: a selective radioligand for PET imaging of brain dopamine transporters

Fluorine-18 labeled 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nort ropane (FECNT) was synthesized in the development of a dopamine transporter (DAT) imaging ligand for positron emission tomography (PET). The methods of radiolabeling and ligand synthesis of FECNT, and the results of the in vitro characterization and in vivo tissue distribution in rats and in vivo PET imaging in rhesus monkeys of [18F]FECNT are described. Fluorine-18 was introduced into 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nort ropane (4) by preparation of 1-[18F]fluoro-2-tosyloxyethane (2) followed by alkylation of 2beta-carbomethoxy-3beta-(4-chlorophenyl)nortropane (3) in 21% radiochemical yield (decay corrected to end of bombardment [EOB]). Competition binding in cells stably expressing the transfected human DAT serotonin transporter (SERT) and norepinephrine transporter (NET) labeled by [3H]WIN 35428, [3H]citalopram, and [3H]nisoxetine, respectively, indicated the following order of DAT affinity: GBR 12909 > CIT > 2beta-carbomethoxy-3beta-(4-chlorophenyl)-8-(3-fluoropropyl) nortropane (FPCT) > FECNT. The affinity of FECNT for SERT and NET was 25- and 156-fold lower, respectively, than for DAT. Blocking studies were performed in rats with a series of transporter-specific agents and demonstrated that the brain uptake of [18F]FECNT was selective and specific for DAT-rich regions. PET brain imaging studies in monkeys demonstrated high [18F]FECNT uptake in the caudate and putamen that resulted in caudate-to-cerebellum and putamen-to-cerebellum ratios of 10.5 at 60 min. [18F]FECNT uptake in the caudate/putamen peaked in less than 75 min and exhibited higher caudate- and putamen-to-cerebellum ratios at transient equilibrium than reported for 11C-WIN 35,428, [11C]CIT/RTI-55, or [18F]beta-CIT-FP. Analysis of monkey arterial plasma samples using high performance liquid chromatography determined that there was no detectable formation of lipophilic radiolabeled metabolites capable of entering the brain. In equilibrium displacement experiments with CIT in rhesus monkeys, radioactivity in the putamen was displaced with an average half-time of 10.2 min. These results indicate that [18F]FECNT is a radioligand that is superior to 11C-WIN 35,428, [11C]CIT/RTI-55, [18F]beta-CIT-FP, and [18F]FPCT for mapping brain DAT in humans using PET.     

Different brain radioactivity curves in a PET study with [11C]beta-CIT labelled in two different positions.

The cocaine congener 2beta-carbomethoxy-3beta-(4'-iodophenyl)tropane (beta-CIT) has a chemical structure that enables labelling with carbon-11 either by N-methylation or by O-methylation. The regional brain uptake of [N-methyl-11C]beta-CIT and [O-methyl-11C]beta-CIT was compared in cynomolgus monkeys using positron emission tomography (PET). The striatal uptake of radioactivity after intravenous injection of [O-methyl-11C]beta-CIT reached a plateau at 30-40 min, whereas the uptake of [N-methyl-11C]beta-CIT increased continuously during the time of the PET measurement. Two of the putative labelled metabolites, [N-methyl-11C]beta-CIT-acid and [O-methyl-11C]nor-beta-CIT were prepared and examined with PET to investigate if they may enter the brain and thus add to the radioactivity uptake obtained with [11C]beta-CIT. Less than 0.4% of injected [N-methyl-11C]beta-CIT-acid entered the brain whereas 5-6% of [O-methyl-11C]nor-beta-CIT entered the brain and accumulated in the striatum and in the thalamus. The fraction of [O-methyl-11C]nor-beta-CIT obtained in plasma after intravenous injection of [O-methyl-11C]nor-beta-CIT, however, never exceeded 3%. Consequently, the formation of [N-methyl-11C]beta-CIT-acid and [O-methyl-11C]nor-beta-CIT cannot be the explanation for the different time-activity curves in the monkey brain demonstrated with [11C]beta-CIT labelled in two different positions. An unidentified labelled lipophilic metabolite, detected in monkey plasma after injection of [O-methyl-11C]beta-CIT, remains as the only possible explanation for the differences between [N-methyl-11C]beta-CIT and [O-methyl-11C]beta-CIT.     

Carbon-11 HOMADAM: a novel PET radiotracer for imaging serotonin transporters

Carbon-11-labeled N,N-dimethyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine (HOMADAM) was synthesized as a new serotonin transporter (SERT) imaging agent. METHODS: Carbon-11 was introduced into HOMADAM by preparation of N-methyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine followed by alkylation with carbon-11 iodomethane. Binding affinities of HOMADAM and the radiolabeling substrate, N-methyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine, were determined in cDNA transfected cells expressing human SERT, dopamine transporters (DAT) and norepinephrine transporters NET using [3H]citalopram, [(125)I]RTI-55 and [3H]nisoxetine, respectively. MicroPET brain imaging was performed in monkeys. Arterial plasma metabolites of HOMADAM were analyzed in a rhesus monkey by high-performance liquid chromatography (HPLC). RESULTS: HOMADAM displayed high affinity for the SERT (Ki = 0.6 nM). N-methyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine displayed moderate affinity for the SERT (Ki = 15.11 nM). The affinities of HOMADAM for the DAT and NET were 2000- and 253-fold lower, respectively, than for the SERT. [11C]HOMADAM was prepared from [11C]iodomethane in approximately 25% radiochemical yield (decay-corrected to end of bombardment). MicroPET brain imaging studies in monkeys demonstrated that [11C]HOMADAM uptake was selectively localized in the midbrain, thalamus, pons, caudate, putamen and medulla. The midbrain-to-cerebellum, pons-to-cerebellum, thalamus-to-cerebellum and putamen-to-cerebellum ratios at 85 min were 4.2, 2.8, 2.3 and 2.0, respectively. HOMADAM binding achieved quasi-equilibrium at 45 min. Radioactivity in the SERT-rich regions of monkey brain was displaceable with R,S-citalopram. Radioactivity in the DAT-rich regions of monkey brain was not displaceable with the DAT ligand RTI-113. Radioactivity in the SERT-rich regions of monkey brain was displaceable with the R,S-reboxetine, a NET ligand with a high nanomolar affinity for SERT. Arterial plasma metabolites of HOMADAM were analyzed in a rhesus monkey by HPLC and displayed a single peak that corresponded to unmetabolized HOMADAM. CONCLUSION: HOMADAM is an excellent candidate for PET primate imaging of brain SERTs.