Comparative evaluation of two serotonin transporter ligands in the human brain: [(11)C](+)McN5652 and [(11)C]cyanoimipramine

Serotonin (5-HT) is considered to be an important transmitter underlying mood and behaviour. Abnormalities of the 5-HT transporter have been suggested in mood disorders, since it is one of the major binding sites of antidepressants. A number of ligands have been developed to visualise the 5-HT transporter in vivo, but only a few have successfully visualised specific binding in vivo. In this study, we comparatively evaluated two ligands for 5-HT transporter, [(11)C](+)McN5652 and [(11)C]cyanoimipramine, in the human brain. Brain uptake of [(11)C](+)McN5652 and [(11)C]cyanoimipramine was measured with PET in 15 healthy volunteers. Second PET scans were performed after pretreatment with the potent 5-HT reuptake inhibitor clomipramine. Data were analysed as regional brain uptake as well as whole brain uptake. In six healthy volunteers uptake of the two ligands was also measured in the lung since it is one of the high-uptake organs in the body. In the brain, high accumulation was observed in the thalamus and striatum, the regions known to contain high densities of 5-HT transporter, for both [(11)C](+)McN5652 and [(11)C]cyanoimipramine. The average ratio of thalamus to cerebellum uptake at 90 min after the tracer injection was approximately 1.6 for [(11)C](+)McN5652 and 1.7 for [(11)C]cyanoimipramine, while the ratios obtained after pretreatment with clomipramine were approximately 1.2. However, the whole brain uptake of [(11)C](+)McN5652 was approximately twice that of [(11)C]cyanoimipramine, while the lung uptake of [(11)C](+)McN5652 was approximately half that of [(11)C]cyanoimipramine. Both [(11)C](+)McN5652 and [(11)C]cyanoimipramine showed sufficient specific binding for performance of a quantitative analysis in the brain. [(11)C](+)McN5652 could be superior because of its higher distribution to the brain.     

Inhibition of serotonin transport by (+)McN5652 is noncompetitive

INTRODUCTION: Imaging of the serotonergic innervation of the brain using positron emission tomography (PET) with the serotonin transporter (SERT) ligand [11C] (+)McN5652 might be affected by serotonin in the synaptic cleft if there is relevant interaction between [11C] (+)McN5652 and serotonin at the SERT. The aim of the present study therefore was to pharmacologically characterize the interaction of [11C] (+)McN5652 and serotonin at the SERT. METHODS: In vitro saturation analyses of [3H]serotonin uptake into HEK293 cells stably expressing the human SERT were performed in the absence and presence of unlabelled (+)McN5652. Data were evaluated assuming Michaelis-Menten kinetics. RESULTS: Unlabelled (+)McN5652 significantly reduced the maximal rate of serotonin transport V(max) of SERT without affecting the Michaelis-Menten constant K(M). CONCLUSIONS: This finding indicates that (+)McN5652 inhibits serotonin transport through the SERT in a noncompetitive manner. This might suggest that [11C] (+)McN5652 PET is not significantly affected by endogenous serotonin.     

In vivo imaging of serotonin transporters with [99mTc]TRODAT-1 in nonhuman primates

[99mTc]TRODAT-1 was the first 99mTc-labeled imaging agent to show specific binding to dopamine transporters (DAT) in the striatum (STR) of human brain. Additionally, in vitro binding and autoradiographic experiments demonstrated that this tracer also binds to serotonin transporters (SERT) in the midbrain/hypothalamus (MB) area. In this study, [99mTc]TRODAT-1 was investigated as a potentially useful ligand to image SERT in the MB of living brain. A total of eight single-photon emission tomography (SPET) scans were performed in two baboons (Papio anubis) after intravenous (i.v.) injection of 740 MBq (20 mCi) of [99mTc]TRODAT-1 using a triple-head gamma camera equipped with ultra-high-resolution fan-beam collimators (scan time: 0-210 min). In four blocking studies, baboons were pretreated with (+)McN5652 (1 mg/kg, i.v.) or methylphenidate (1 mg/kg, i.v.) to specifically block SERT or DAT, respectively. After co-registration with magnetic resonance images of the same baboon, a region of interest analysis was performed using predefined templates to calculate specific uptake in the midbrain area and the striatum, with the cerebellum as the background region [(MB-CB)/CB, (STR-CB)/CB]. Additionally, two PET scans of the same baboons were performed after i.v. injections of 74-111 MBq (2-3 mCi) of [11C](+)McN5652 to identify the SERT sites. In [99mTc]TRODAT-1/SPET scans, the SERT sites in the MB region were clearly visualized. Semiquantitative analysis revealed a specific uptake in MB ([MB-CB]/CB) of 0.30+/-0.02, which was decreased to 0. 040+/-0.005 after pretreatment with nonradioactive (+)McN5652, a selective SERT ligand. Pretreatment with methylphenidate reduced the specific binding of [99mTc]TRODAT-1 to DAT sites [(STR-CB)/CB] from 2.45+/-0.13 to 0.32+/-0.04 without any effect on its binding to SERT sites [(MB-CB)/CB], which was confirmed by the co-registration of the [11C](+)McN5652/PET scans. This preliminary study suggests that specific binding of [99mTc]TRODAT-1 to SERT sites can be detected by in vivo SPET imaging despite the low target to background ratio. These findings provide impetus for further development of similar compounds with improved binding affinity and selectivity to SERT sites.     

Single-photon emission tomography imaging of serotonin transporters in the nonhuman primate brain with [(123)I]ODAM.

We have described previously a selective serotonin transporter (SERT) radioligand, [(123)I]IDAM. We now report a similarly potent, but more stable IDAM derivative, 5-iodo-2-[2-[(dimethylamino)methyl]phenoxy]benzyl alcohol ([(123)I]ODAM). The imaging characteristics of this radioligand were studied and compared against [(123)I]IDAM. Dynamic sequences of single-photon emission tomography (SPET) scans were obtained on three female baboons after injection of 375 MBq of [(123)I]ODAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 120 min after injection of [(123)I]ODAM. Total integrated brain uptake of [(123)I]ODAM was about 30% higher than [(123)I]IDAM. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT. Peak specific binding in the midbrain occurred 120 min after injection, with an equilibrium midbrain to cerebellar ratio of 1. 50+/-0.08, which was slightly lower than the value for [(123)I]IDAM (1.80+/- 0.13). Both the binding kinetics and the metabolism of [(123)I]ODAM were slower than those of [(123)I]IDAM. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited washout from areas with high concentrations of SERT, with a dissociation kinetic rate constant k(off)=0.0085+/-0.0028 min(-1) in the midbrain. Similar studies using nisoxetine and methylphenidate showed no displacement, consistent with its low binding affinity to norepinephrine and dopamine transporters, respectively. These results suggest that [(123)I]ODAM is suitable for selective SPET imaging of SERT in the primate brain, with higher uptake and slower kinetics and metabolism than [(123)I]IDAM, but also a slightly lower selectivity for SERT.     

In vivo imaging of serotonin transporters with [99mTc]TRODAT-1 in nonhuman primates

[^99mTc]TRODAT-1 was the first ^99mTc-labeled imaging agent to show specific binding to dopamine transporters (DAT) in the striatum (STR) of human brain. Additionally, in vitro binding and autoradiographic experiments demonstrated that this tracer also binds to serotonin transporters (SERT) in the midbrain/hypothalamus (MB) area. In this study, [^99mTc]TRODAT-1 was investigated as a potentially useful ligand to image SERT in the MB of living brain. A total of eight single-photon emission tomography (SPET) scans were performed in two baboons (Papio anubis) after intravenous (i.v.) injection of 740 MBq (20 mCi) of [^99mTc]TRODAT-1 using a triple-head gamma camera equipped with ultra-high-resolution fan-beam collimators (scan time: 0–210 min). In four blocking studies, baboons were pretreated with (+)McN5652 (1 mg/kg, i.v.) or methylphenidate (1 mg/kg, i.v.) to specifically block SERT or DAT, respectively. After co-registration with magnetic resonance images of the same baboon, a region of interest analysis was performed using predefined templates to calculate specific uptake in the midbrain area and the striatum, with the cerebellum as the background region [(MB–CB)/CB, (STR–CB)/CB]. Additionally, two PET scans of the same baboons were performed after i.v. injections of 74–111 MBq (2–3 mCi) of [¹¹C](+)McN5652 to identify the SERT sites. In [^99mTc]TRODAT-1/SPET scans, the SERT sites in the MB region were clearly visualized. Semiquantitative analysis revealed a specific uptake in MB ([MB–CB]/CB) of 0.30±0.02, which was decreased to 0.040±0.005 after pretreatment with nonradioactive (+)McN5652, a selective SERT ligand. Pretreatment with methylphenidate reduced the specific binding of [^99mTc]TRODAT-1 to DAT sites [(STR-CB)/CB] from 2.45±0.13 to 0.32±0.04 without any effect on its binding to SERT sites [(MB–CB)/CB], which was confirmed by the co-registration of the [¹¹C](+)McN5652/PET scans. This preliminary study suggests that specific binding of [^99mTc]TRODAT-1 to SERT sites can be detected by in vivo SPET imaging despite the low target to background ratio. These findings provide impetus for further development of similar compounds with improved binding affinity and selectivity to SERT sites. Received 15 September and in revised form 15 November 1998     

Comparative evaluation of serotonin transporter radioligands 11C-DASB and 11C-McN 5652 in healthy humans.

Alterations of serotonin transporters (SERT) are implicated in a large number of psychiatric conditions. (11)C-(+)-6beta-(4-Methylthiophenyl)-1,2,3,5,6alpha,10beta-hexahydropyrrolo[2,1-a]isoquinoline ((11)C-McN 5652) was the first PET radiotracer successfully developed as a SERT imaging agent. Recently, (11)C-3-amino-4-(2-dimethylaminomethylphenylthio)benzonitrile ((11)C-DASB) was introduced as an alternative to (11)C-McN 5652. Comparative evaluation of (11)C-DASB and (11)C-McN 5652 in baboons indicates that (11)C-DASB is associated with (a) lower nonspecific binding in the brain, (b) higher plasma free fraction, and (c) faster plasma clearance and brain uptake kinetics, enabling measurement of SERT parameters in a shorter scanning time. The purpose of this study was to compare these 2 agents in healthy humans. METHODS: Six healthy volunteers underwent 2 PET scans on the same day, one with (11)C-DASB and one with (11)C-McN 5652, in counterbalanced order. Regional distribution volumes (V(T)) were derived for 16 brain regions by kinetic analysis using the arterial input function. RESULTS: Both (11)C-DASB and (11)C-McN 5652 displayed similar patterns of accumulation: highest levels in the midbrain, thalamus and striatum; intermediate in the limbic regions; low in the neocortex; and lowest in the cerebellum. (11)C-DASB cerebellar V(T) (10.1 +/- 2.0 mL g(-1)) was lower than that of (11)C-McN 5652 (20.8 +/- 3.6 mL g(-1)), indicating lower nonspecific binding. As a result, regional specific-to-nonspecific equilibrium partition coefficients (V(3)") of (11)C-DASB were higher compared with those of (11)C-McN 5652 (for example, midbrain V(3)" of (11)C-DASB and (11)C-McN 5652 were 2.04 +/- 0.44 and 1.20 +/- 0.34, respectively). The plasma free fraction was 8.9% +/- 1.6% for (11)C-DASB and was not measurable for (11)C-McN 5652. In contrast to the situation observed in baboons, plasma clearances of both compounds were similar in humans, and the minimal scanning times required to derive time-invariant distribution volumes in all regions were comparable for both tracers (95 min). CONCLUSION: With the exception of the scanning time, predictions from baboon studies were confirmed in humans. The higher specific-to-nonspecific ratios of (11)C-DASB are a critical advantage. This property will be especially important for the measurement of SERT in regions with moderate density, such as the limbic regions, where alterations of serotonin transmission might be associated with anxiety and depression.     

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:     

Effects of smoking on the lung accumulation of [<Superscript>11</Superscript>C]McN5652

Objective The lung is one of the key organs for determining the distribution of drugs in the human body. Various factors influence the accumulation of drugs. In this study, we investigated the effects of smoking on drug distribution to the lung using radiolabeled drugs. Methods We measured the lung uptake of [¹¹C](+)McN5652, a radioligand for serotonin transporter (5-HTT), and inactive enantiomer [¹¹C](−)McN5652 in 19 healthy men (12 nonsmokers and 7 smokers) using positron emission tomography. Pretreatment study was performed by the administration of clomipramine (50 mg), a potent 5-HTT inhibitor. Results The mean lung uptake of [¹¹C](+)McN5652 and [¹¹C](−)McN5652 was significantly higher in smokers than in nonsmokers. The lung uptake of [¹¹C](+)McN5652 decreased after pretreatment with clomipramine, whereas that of [¹¹C](−)McN5652 was not affected by clomipramine. Conclusions Lung uptake of [¹¹C](−)McN5652 was influenced by smoking, possibly because the probable nonspecific binding accumulation was changed as [¹¹C](−)McN5652 was reported to have negligible affinity to 5-HTT. Smoking might be one of the important factors when distribution of radioligands is considered.     

Comparison of (+)-(11)C-McN5652 and (11)C-DASB as serotonin transporter radioligands under various experimental conditions.

There has been considerable interest in the development of a PET radioligand selective for the serotonin (5-hydroxytryptamine [5-HT]) transporter (SERT) that can be used to image 5-HT neurons in the living human brain. The most widely used SERT radiotracer to date, trans-1,2,3,5,6,10-beta-hexahydro-6-[4-(methylthio)phenyl[pyrrolo-[2,1-a]isoquinoline ((+)-(11)C-McN5652), has been successful in this regard but may have some limitations. Recently, another promising SERT radiotracer, 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile ((11)C-DASB), has been described. The purpose of this study was to compare and contrast (+)-(11)C-McN5652 and (11)C-DASB under various experimental conditions. METHODS: Radioligand comparisons were performed in a control baboon, a baboon with reduced SERT density ((+/-)-3,4-methylenedioxymethamphetamine [MDMA] lesion), and a baboon with reduced SERT availability (paroxetine pretreatment). Under each of these experimental conditions, repeated (triplicate) PET studies were performed with each ligand. RESULTS: Both radiotracers bound preferentially in brain regions known to contain high SERT density. For both ligands, there was a high correlation between the amount of regional brain ligand binding and the known regional brain concentration of SERT. Binding of both ligands was decreased after MDMA neurotoxicity (reduced SERT density), and (+)-(11)C-McN5652 and (11)C-DASB were comparably effective in detecting reduced SERT density after MDMA-induced 5-HT neurotoxicity. Pretreatment with paroxetine dramatically altered the metabolism and kinetics of both tracers and appeared to displace both ligands primarily from regions with high SERT density. Compared with (+)-(11)C-McN5652, (11)C-DASB had higher brain activity and a faster washout rate and provided greater contrast between subcortical and cortical brain regions. CONCLUSION: (11)C-DASB and (+)-(11)C-McN5652 are suitable as PET ligands of the SERT and for detecting MDMA-induced 5-HT neurotoxicity. (11)C-DASB may offer some advantages. Additional studies are needed to further characterize the properties and capabilities of both ligands in health and disease.     

Single-photon emission tomography imaging of serotonin transporters in the nonhuman primate brain with [123I]ODAM

We have described previously a selective serotonin transporter (SERT) radioligand, [¹²³I]IDAM. We now report a similarly potent, but more stable IDAM derivative, 5-iodo-2-[2-[(dimethylamino)methyl]phenoxy]benzyl alcohol ([¹²³I]ODAM). The imaging characteristics of this radioligand were studied and compared against [¹²³I]IDAM. Dynamic sequences of single-photon emission tomography (SPET) scans were obtained on three female baboons after injection of 375 MBq of [¹²³I]ODAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 120 min after injection of [¹²³I]ODAM. Total integrated brain uptake of [¹²³I]ODAM was about 30% higher than [¹²³I]IDAM. After 60–120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT. Peak specific binding in the midbrain occurred 120 min after injection, with an equilibrium midbrain to cerebellar ratio of 1.50±0.08, which was slightly lower than the value for [¹²³I]IDAM (1.80± 0.13). Both the binding kinetics and the metabolism of [¹²³I]ODAM were slower than those of [¹²³I]IDAM. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited washout from areas with high concentrations of SERT, with a dissociation kinetic rate constant k _off=0.0085±0.0028 min^–1 in the midbrain. Similar studies using nisoxetine and methylphenidate showed no displacement, consistent with its low binding affinity to norepinephrine and dopamine transporters, respectively. These results suggest that [¹²³I]ODAM is suitable for selective SPET imaging of SERT in the primate brain, with higher uptake and slower kinetics and metabolism than [¹²³I]IDAM, but also a slightly lower selectivity for SERT. Received 1 May and in revised form 31 May 1999     

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.     

Single-photon emission tomography imaging of serotonin transporters in the non-human primate brain with the selective radioligand [(123)I]IDAM.

A new radioligand, 5-iodo-2-[[2-2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol ([(123)I]IDAM), has been developed for selective single-photon emission tomography (SPET) imaging of SERT. In vitro binding studies suggest a high selectivity of IDAM for SERT (K(i)=0.097 nM), with considerably lower affinities for norepinephrine and dopamine transporters (NET K(i)= 234 nM and DAT K(i)>10 microM, respectively). In this study the biodistribution of SERT in the baboon brain was investigated in vivo using [(123)I]IDAM and SPET imaging. Dynamic sequences of SPET scans were performed on three female baboons (Papio anubis) after injection of 555 MBq of [(123)I]IDAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 90-120 min after injection of [(123)I]IDAM. Similar studies were performed using a NET inhibitor, nisoxetine, and a DAT blocker, methylphenidate. After 60-120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT, with the highest uptake in the midbrain area (hypothalamus, raphe nucleus, substantia nigra), and the lowest uptake in the cerebellum (an area presumed free of SERT). Peak specific binding in the midbrain occurred at 120 min, with a ratio to the cerebellum of 1.80+/-0.13. At 30 min, 85% of the radioactivity in the blood was metabolite. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited rapid washout from areas with high concentrations of SERT (dissociation rate constant in the midbrain, averaged over three baboons, k(off)=0. 025+/-0.002 min(-1)), while the cerebellar activity distribution was undisturbed (washout rate 0.0059+/- 0.0003 min(-1)). Calculation of tracer washout rate pixel-by-pixel enabled the generation of parametric images of the dissociation rate constant. Similar studies using nisoxetine and methylphenidate had no effect on the distribution of [(123)I]IDAM in the brain. These results suggest that [(123)I]IDAM is suitable for selective SPET imaging of SERT in the primate brain, with high contrast, favorable kinetics, and negligible binding to either NET or DAT.     

Serotonin transporters in the midbrain of Parkinson's disease patients: a study with 123I-beta-CIT SPECT.

In Parkinson's disease (PD), both neuropathologic and biochemical studies suggest that serotonin (5-hydroxytryptamine [5-HT]) neurons are affected by the disease process. The integrity of 5-HT transporters was assessed in PD patients with SPECT using 2beta-carbomethoxy-3beta-(4-(123)I-iodophenyl)tropane ((123)I-beta-CIT), which binds with high affinity to both dopamine (DA) and 5-HT transporters. METHODS: Forty-five PD patients at relatively early stages (mean Hoehn-Yahr stage, 2.0 +/- 0.7; range, 1-3) and 7 age-matched healthy control subjects had 15 scans over a 24-h period after injection of (123)I-beta-CIT using a 3-head SPECT system. In the midbrain, the 5-HT transporter parameter k(3)/k(4) was estimated by 3 noninvasive methods: pseudoequilibrium ratio (R(PE)) method, area ratio (R(A)) method, and a modified graphic method that derives the ratio of ligand distribution volumes (R(V)). Striatal V(3)", the DA transporter parameter that is equivalent to k(3)/k(4), was measured using the images acquired at 24 h after tracer injection. All measures were derived using the cerebellum as the reference region. RESULTS: In control subjects, the (123)I-beta-CIT activity in the midbrain reached a peak at 91 +/- 21 min after injection and then washed out at a slow rate (1.1%/h +/- 0.5%/h). The peak specific uptake in the midbrain occurred at 315 +/- 46 min. In PD patients, the temporal patterns of the midbrain and cerebellar activity were not significantly different from those in control subjects. None of midbrain R(PE), R(A), and R(V) was significantly different between control subjects and PD patients, whereas striatal V(3)" was bilaterally reduced in all patients, being 32% lower than that of the control subjects (P = 0.002). In PD patients, none of the midbrain outcome measures was significantly correlated with either striatal V(3)" or motor or nonmotor symptom ratings, including the Hoehn-Yahr stage and the Unified Parkinson's Disease Rating Scale scores. When the studies of 7 PD patients with depression were analyzed separately, none of the midbrain outcome measures in these patients either was different significantly from control values or correlated with the Hamilton Depression Rating Scale score. CONCLUSION: These results suggest that DA and 5-HT transporters are differentially affected in PD, and 5-HT transporters in the midbrain region may not be affected in relatively early stages of PD. Alternatively, 5-HT transporters in the remaining neurons may be upregulated, thus raising the midbrain 5-HT transporter density to almost normal levels.     

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.     

Single-photon emission tomography imaging of serotonin transporters in the non-human primate brain with the selective radioligand [123I]IDAM

A new radioligand, 5-iodo-2-[[2–2-[(dimethylamino)methyl]phenyl]thio]benzyl alcohol ([¹²³I]IDAM), has been developed for selective single-photon emission tomography (SPET) imaging of SERT. In vitro binding studies suggest a high selectivity of IDAM for SERT (K _i=0.097 nM), with considerably lower affinities for norepinephrine and dopamine transporters (NET K _i= 234 nM and DAT K _i>10 μM, respectively). In this study the biodistribution of SERT in the baboon brain was investigated in vivo using [¹²³I]IDAM and SPET imaging. Dynamic sequences of SPET scans were performed on three female baboons (Papio anubis) after injection of 555 MBq of [¹²³I]IDAM. Displacing doses (1 mg/kg) of the selective SERT ligand (+)McN5652 were administered 90–120 min after injection of [¹²³I]IDAM. Similar studies were performed using a NET inhibitor, nisoxetine, and a DAT blocker, methylphenidate. After 60–120 min, the regional distribution of tracer within the brain reflected the characteristic distribution of SERT, with the highest uptake in the midbrain area (hypothalamus, raphe nucleus, substantia nigra), and the lowest uptake in the cerebellum (an area presumed free of SERT). Peak specific binding in the midbrain occurred at 120 min, with a ratio to the cerebellum of 1.80±0.13. At 30 min, 85% of the radioactivity in the blood was metabolite. Following injection of a competing SERT ligand, (+)McN5652, the tracer exhibited rapid washout from areas with high concentrations of SERT (dissociation rate constant in the midbrain, averaged over three baboons, k _off=0.025±0.002 min^–1), while the cerebellar activity distribution was undisturbed (washout rate 0.0059± 0.0003 min^–1). Calculation of tracer washout rate pixel-by-pixel enabled the generation of parametric images of the dissociation rate constant. Similar studies using nisoxetine and methylphenidate had no effect on the distribution of [¹²³I]IDAM in the brain. These results suggest that [¹²³I]IDAM is suitable for selective SPET imaging of SERT in the primate brain, with high contrast, favorable kinetics, and negligible binding to either NET or DAT. Received: 1 February and in revised form 2 March 1999     

N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)-benzylamine (4-(18)F-ADAM): an improved PET radioligand for serotonin transporters.

There has been considerable interest in the development of PET radioligands that are useful for imaging serotonin transporter (SERT) in the living human brain. For the last decade, (11)C-(+)McN5652 has been the most promising PET agent for studying SERT in humans. However, this agent has some limitations. Recently, a new promising SERT PET radioligand, 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile, has been reported. We recently reported the synthesis of a new (18)F-labeled SERT PET radioligand, N,N-dimethyl-2-(2-amino-4-(18)F-fluorophenylthio)benzylamine (4-(18)F-ADAM), which may have advantages over (11)C-labeled radioligands. The purpose of this study was to evaluate this newly developed (18)F-labeled PET radioligand as a promising agent for studying SERT in the living human brain. METHODS: This agent was evaluated by studying its in vitro binding to different monoamine transporters, its in vivo biodistributions in rats, its integrity and pharmacologic profiles in rat brain, and its distribution in a female baboon brain. RESULTS: In vitro binding assays showed that 4-F-ADAM displayed high affinity to SERT sites (inhibition constant = 0.081 nmol/L, using membrane preparations of LLC-PK1 cells expressing the specific transporter) and showed more than 1,000- and 28,000-fold selectivity for SERT over norepinephrine transporter and dopamine transporter, respectively. Biodistribution of 4-(18)F-ADAM in rats showed a high initial uptake and slow clearance in the brain (2.13%, 1.90%, and 0.95% injected dose per organ at 2, 30, and 60 min after intravenous injection, respectively), with the specific binding peaking at 2 h after injection (hypothalamus/cerebellum = 12.49). The uptake in blood, muscle, lung, kidney, and liver was also initially high but cleared rapidly. The radioactivity in the femur increases with time for 4-(18)F-ADAM, indicating that in vivo defluorination may occur. In vivo metabolism studies in rats showed that 4-(18)F-ADAM was not metabolized in rat brain (>96% of radioactivity was recovered as parent compound at 1 h after injection). However, it metabolized rapidly in the blood. Less than 7% of the radioactivity recovered from plasma was the parent compound, with the majority of radioactivity in the plasma not extractable by ethyl acetate. Blocking studies showed significant decreases in the uptake of 4-(18)F-ADAM in the brain regions (hypothalamus, hippocampus, and striatum) where SERT concentrations are high when rats were pretreated with (+)McN5652 (2 mg/kg 5 min before intravenous injection of 4-(18)F-ADAM). However, changes in the uptake of 4-(18)F-ADAM in these brain regions were less significant when rats were pretreated with either methylphenidate or nisoxetine. The baboon study showed that uptake of 4-(18)F-ADAM in the midbrain peaked at approximately 1 h after injection and then declined slowly. The ratios of the radioactivity in the midbrain to that in the cerebellum (where the concentration of SERT is low) at 2 and 3 h after injection were 3.2 and 4.2, respectively. CONCLUSION: 4-(18)F-ADAM is suitable as a PET radioligand for studying SERT in the living brain. Further characterization of this new radioligand in humans is warranted.     

Synthesis and evaluation of N,N-dimethyl-2-(2-amino-5-[F]fluorophenylthio)benzylamine (5-[F]-ADAM) as a serotonin transporter imaging agent

The synthesis and evaluation of a new serotonin transporter (SERT) imaging agent, N,N-dimethyl-2-(2-amino-5-[¹⁸F]fluorophenylthio)benzylamine (5-[¹⁸F]-ADAM) is reported. Nucleophilic substitution of N,N-dimethyl-2-(2-nitro-5-bromophenylthio)benzylamine with K[¹⁸F]/Kryptofix 2.2.2 in DMSO at 125°C followed by reduction with NaBH₄–Cu(OAc)₂ in EtOH at 78°C and purification with HPLC produces the desired compound with an unoptimized yield of 5–10% in a synthesis time of 150 min from EOB. The biodistribution of 5-[¹⁸F]-ADAM in rats showed a high initial uptake and relatively rapid clearance in the brain (3.221±0.762, 0.440±0.059, 0.160±0.035 and 0.028±0.003% injected dose/organ at 2, 30, 60 and 120 min after I.V. injection, respectively) with the specific binding peaking at 1 h postinjection (hypothalamus/cerebellum and hippocampus/cerebellum were 2.97 and 3.59, respectively). The initial uptake in blood, lung, kidney and heart were also high, but it cleared rapidly. The radioactivity in the femur increased with time for 5-[¹⁸F]-ADAM indicating that in vivo defluorination may occur. Metabolism studies in rats showed that 5-[¹⁸F]-ADAM was not metabolized in rat brain, but was metabolized rapidly in the blood. Blocking experiments showed that there were significant decreases in the uptake of 5-[¹⁸F]-ADAM in the brain regions (hypothalamus, hippocampus and striatum) where SERT concentrations are high when rats were pretreated with (+)McN 5652 (2 mg/kg, 5 min prior to IV injection of 5-[¹⁸F]-ADAM). These results suggest that 5-[¹⁸F]-ADAM may be a potential new serotonin transporter PET imaging agent. However, due to its rapid wash-out from the brain, defluorination in vivo and lower uptake in the brain than 4-[¹⁸F]-ADAM, 5-[¹⁸F]-ADAM may not be as useful as 4-[¹⁸F]-ADAM as a SERT imaging agent.     

Synthesis of S-([18F]fluoromethyl)-(+)-McN5652 as a potential PET radioligand for the serotonin transporter

The present study describes the synthesis of the [18F]fluoromethyl analogue of (+)-McN5652 ([18F]FMe-McN) as a new potential tracer for the serotonin transporter. In vitro binding studies have shown that FMe-McN displays only slightly lower affinity for the serotonin transporter (K(i) = 2.3 +/- 0.1 nM) than (+)-McN5652 (K(i) = 0.72 +/- 0.2 nM). The radiofluorinated tracer [18F]FMe-McN was prepared by reaction of normethyl (+)-McN5652 with the fluoromethylation agent [18F]bromofluoromethane in an overall radiochemical yield of 5 +/- 1% (decay-corrected, related to [18F]fluoride) and with high specific radioactivity (200-2,000 GBq/micromol at the end of synthesis).     

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.     

Is correction for age necessary in SPECT or PET of the central serotonin transporter in young, healthy adults?

PET and SPECT have suggested that there is an age-related decline of up to 10% per decade in the availability of brain serotonin transporter (SERT) in healthy subjects, starting as early as the age of 20 y. The aim of the present study was to verify these findings in young subjects. METHODS: The equilibrium specific-to-nonspecific partition coefficient V'(3) of the SERT ligand (11)C-(+)McN5652 was obtained for 29 healthy subjects aged 18-33 y. V'(3) was tested for age dependence by linear regression analysis using both a volumes-of-interest approach and voxel-based statistical parametric mapping. The sex of the subject and the season of year were considered nuisance variables. RESULTS: Age had no significant effect on V'(3). The power for the detection of an age-related decline in V'(3) of the magnitude reported previously was 0.917. CONCLUSION: These findings indicate that age is not a relevant confounding factor for SERT availability as measured by (11)C-(+)McN5652 PET in healthy adults up to the age of about 35 y.